# Two aspects of longevity are associated with rates of loss of telomeres in birds

F. Stephen Dobson, Quentin Schull, François Criscuolo

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F. Stephen Dobson, Quentin Schull, François Criscuolo. Two aspects of longevity are associated with rates of loss of telomeres in birds. Ecology and Evolution, 2022, 12 (10), 10.1002/ece3.9364. hal-03859962

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# Two aspects of longevity are associated with rates of loss of telomeres in birds

F. Stephen Dobson, Quentin Schull, François Criscuolo

► To cite this version:

F. Stephen Dobson, Quentin Schull, François Criscuolo. Two aspects of longevity are associated with rates of loss of telomeres in birds. Ecology and Evolution, 2022, 12 (10), 10.1002/ece3.9364. hal-03859962v2

HAL Id: hal-03859962

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Submitted on 17 Jan 2025

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archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

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<figure><img src="image_1.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
Two aspects of longevity are associated with rates of loss of telomeres in birds

F.S. Dobson <fnref n="1" />,<fnref n="2" />, Q. Schull <fnref n="1" />,<fnref n="3" />, and F. Criscuolo <fnref n="1" />

Key words: body size, longevity, telomere dynamics, MCMCglmm, phylogenetic correlation

Corresponding author: F.S. Dobson, fsdobson@msn.com

<footnote n="1">¹ University of Strasbourg, CNRS, Institut Pluridisciplinaire Hubert Curien, UMR 7178, 67000 Strasbourg, France</footnote>

<footnote n="2">² Department of Biological Sciences, Auburn University, Auburn, AL, USA</footnote>

<footnote n="3">³ present affiliation: MARBEC, University of Montpellier, IFREMER, IRD, CNRS, Sète, France</footnote>
## RESEARCH ARTICLE

Ecology and Evolution

Open Access

WILEY

# Two aspects of longevity are associated with rates of loss of telomeres in birds

F. Stephen Dobson<fnref n="1" />,<fnref n="2" /> ID

Quentin Schull<fnref n="1" />,<fnref n="3" />

François Criscuolo<fnref n="1" /> ID

### Correspondence

F. Stephen Dobson, CNRS, Institut Pluridisciplinaire Hubert Curien, UMR 7178, University of Strasbourg, 67000 Strasbourg, France.

Email: fsdobson@msn.com

### Funding information

Agence Nationale de la Recherche, Grant/Award Number: 234061; Région Grand Est and the Eurométropole de Strasbourg; Université de Strasbourg Institut d'Etudes Avancées

### Abstract

Telomeres, the terminal repetitive DNA sequences at the ends of linear chromosomes, have strong associations with longevity in some major taxa. Longevity has been linked to rate of decline in telomere length in birds and mammals, and absolute telomere length seems to be associated with body mass in mammals. Using a phylogenetic comparative method and 30 species of birds, we examined longevity (reflected by maximum lifespan), absolute telomere length, the rate of change in telomere length (TROC), and body mass (often strongly associated with longevity) to ascertain their degree of association. We divided lifespan into two life-history components, one reflected by body size (measured as body mass) and a component that was statistically independent of body mass. While both lifespan and body mass were strongly associated with a family tree of the species (viz., the phylogeny of the species), telomere measures were not. Telomere length was not significantly associated with longevity or body mass or our measure of mass-independent lifespan. TROC, however, was strongly associated with mass-independent lifespan, but only to a much lesser degree at best with body mass-predicted lifespan. Our results supported an association of TROC and longevity, in particular longevity that was independent of body size and part of the pace-of-life syndrome of life histories.

### KEYWORDS

body size, longevity, MCMCglmm, phylogenetic correlation, telomere dynamics

### TAXONOMY CLASSIFICATION

Ecological genetics

## 1 | INTRODUCTION

Telomeres are repetitive nucleotide sequences at the terminal regions of eukaryote chromosomes. They serve to protect healthy chromosomes from DNA repair mechanisms that otherwise act on the terminal ends of chromosomes and organize the replication of DNA during cell division (de Lange, 2009). During replication,

telomere sequences may be lost, thus shortening the telomere end (Olonikov, 1973). Telomerase may replace the lost sequences, thus lengthening it (Chan & Blackburn, 2004). Nonetheless, cells with over-shortened telomeres become “senescent” or “self-destruct” (termed apoptosis). When apoptosis occurs, the DNA-encoded information of the cell is removed from the organism (Blackburn, 2000). Thus, telomeres may play a role in both cell

This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

© 2022 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd.

<figure><img src="image_2.png" /></figure>

<footnote n="1">¹University of Strasbourg, Institut Pluridisciplinaire Hubert Curien, UMR 7178, CNRS, Strasbourg, France</footnote>

<footnote n="2">²Department of Biological Sciences, Auburn University, Auburn, Alabama, USA</footnote>

<footnote n="3">³MARBEC, University of Montpellier, IFREMER, IRD, CNRS, Sète, France</footnote>
ABSTRACT

Telomeres, the terminal repetitive DNA sequences at the ends of linear chromosomes, have strong associations with longevity in some major taxa. Longevity has been linked to rate of decline in telomere length in birds and mammals, and absolute telomere length seems to be associated with body mass in mammals. Using a phylogenetic comparative method and 30 species of birds, we examined longevity (reflected by maximum lifespan), absolute telomere length, the rate of change in telomere length (TROC), and body mass (often strongly associated with longevity) to ascertain their degree of association. We divided lifespan into two life-history components, one reflected by body size (measured as body mass), and a component that was statistically independent of body mass. While both lifespan and body mass were strongly associated with a family tree of the species (viz., the phylogeny of the species), telomere measures were not. Telomere length was not significantly associated with longevity or body mass, or our measure of mass-independent lifespan. TROC, however, was strongly associated with mass-independent lifespan, but only to a much lesser degree at best with body mass-predicted lifespan. Our results supported an association of TROC and longevity, in particular longevity that was independent of body size and part of the pace-of-life syndrome of life histories.
senescence (or alternatively in cell immortalization due to the activ-
ity of telomerase; Tian et al., 2018), accumulation of senescent cells
in organs (Campisi, 2005), and organismal senescence (Blasco, 2007;
Young, 2018). Further, accelerated telomere loss at a given chrono-
logical age may indicate decreasing organismal condition, especially
during early development, and may underlie both physiological
stress and a shorter life (viz., advanced senescence; Bize et al., 2009;
Boonekamp et al., 2014; Haussmann et al., 2003; Pepper et al., 2018;
Sheldon et al., 2021; Suyka et al., 2016; Whittemore et al., 2019).

Previous studies have suggested that due to the association of
rate of telomere loss from chromosomes and organismal senescence,
telomere dynamics during life are closely and functionally associated
with lifespan among species of different body sizes and pace of life
(e.g., Dantzer & Fletcher, 2015; Tricola et al., 2018). These studies
primarily focused on birds and recognized the possible importance
of body size and historical patterns (viz., the influence of phylogeny)
in explaining a general pattern of relatively slowed loss of telomeres
during life in larger species with syndromes of slower paces of life.
These studies applied phylogenetic comparisons to 14 and 19 species
of birds (TRF data), respectively, relatively small samples for robust
phylogenetic analyses (though Dantzer & Fletcher, 2015 presented
an analysis that assumed no significant phylogenetic associations).
Tricola et al. (2018) suggested that the rate of telomere shortening
exhibited a strong historical pattern that may have coevolved with
lifespan. Both studies concluded that larger species with relatively
slower life histories exhibited reduced rates of telomere shortening.

These key previous studies raised a series of questions that
might be examined with a larger sample of species of birds. First,
how flexible are telomere traits over phylogenetic history? Tricola
et al. (2018) found that telomere length was not strongly influenced
by the phylogenetic pattern, but telomere rate of change (TROC)
was. Alternatively, Criscuolo et al. (2021) found that neither adult
telomere length nor TROC showed a strong phylogenetic pattern
in a corrected sample size of 52 bird species, a different result that
needs to be explained.

Second, how do longevity, body size, and the pace of life interact
with telomere dynamics? Dantzer and Fletcher (2015) found that all
three variables covaried strongly with TROC, and TROC was lower
for the longest-lived bird species. The latter result was confirmed
by Tricola et al. (2018) with or without body mass used as a covari-
ate. In a phylogenetic comparative analysis of nine species of birds
and mammals, Le Pepke and Eisenberg (2020) reported a low rate of
telomere loss in long-lived species but a trivial effect of body mass.
These studies took a phylogenetic comparative approach where
regressions were used to "control" for the phylogenetic patterns
underlying variables. Still, none of them quantified how those phy-
logenetically controlled patterns of telomere dynamics change with
lifespan at a given body size, that is, when influences of body size are
statistically controlled (Udroiu, 2020).

Third, does telomere length per se change with species lifes-
pan, with or without the influences of body size taken into ac-
count? Gomes et al. (2011) studied 61 species of mammals and
found that adult telomeres were shorter in the longest-lived

species. Both body mass and longevity showed significant neg-
ative associations with telomerase activity and telomere length,
respectively, independent of the phylogenetic pattern among the
species (Gorbunova & Seluanov, 2009). The conclusion of Gomes
et al. (2011) was that, for large species to evolve long lifespans,
replicative aging occurred (i.e., short telomeres combined with re-
pressed telomerase activity). A result confirmed by a re-analysis
of the same dataset recently (Le Pepke & Eisenberg, 2021), with
emphasis on an inverse association of telomere length with body
mass. These results suggest that short telomeres may have co-
evolved in long-lived species as a consequence of body size, un-
derlying the necessity for large species to control for higher risks
of cell immortalization by a widespread cellular mechanism (Tian
et al., 2018). Interestingly, the phylogeny-adjusted analyses of
Tricola et al. (2018) conducted in birds found no significant associ-
ation of telomere length and longevity, whether or not influences
of body mass were included. However, longevity and body mass
are known to show strong covariance (Dantzer & Fletcher, 2015)
and thus may be collinear. The analyses of more bird species by
Criscuolo et al. (2021), however, found no significant association of
adult telomere length and either body size or the pace of life, with
or without inclusion of the phylogenetic pattern in the analyses,
though they did not specifically examine longevity and its single-
trait association with telomere length.

The purpose of our present study was to examine associations of
telomere length and TROC on the one hand, and body size and lon-
gevity on the other hand in birds. We did this using a restricted sam-
ple size of 30 of the bird species (see below for justification) reviewed
in the comparative analysis of Criscuolo et al. (2021). We first asked
whether any of the variables showed evidence of strong phylogenetic
pattern, using the Bayesian meta-analysis approach of Hadfield and
Nakagawa (2010). Longevity and body size are closely associated in
birds (e.g., Bennett & Owens, 2002; Criscuolo et al., 2021; Dantzer &
Fletcher, 2015). Larger species reflect many aspects of life histories
that covary over evolutionary time, in part because it takes longer to
grow and survive to a large body size (Dobson, 2007). Further, bird
species vary along a "slow-fast continuum" that reflects alternative
paces of life that are independent of body size (Gaillard et al., 1989).
Thus, we examined variation in longevity that was strongly associ-
ated with body size ("mass-predicted lifespan"), and longevity that
was statistically independent of body size ("mass-independent lifes-
pan"). The latter reflects changes in lifespan that can be described
as varying with the pace of life (Criscuolo et al., 2021; Dobson &
Oli, 2007). Because differences between phylogenetically adjusted
and unadjusted associations of traits can be biologically informative
(Price, 1997), we compared both of the aspects of lifespan to telo-
more length and TROC.

We also addressed a further issue with respect to how TROC is
measured. First, our sample included estimates of TROC that used
mean differences in telomere lengths between chicks and older birds
(after Criscuolo et al., 2021). Such estimates have the advantage of
including the chick period, when the greatest rates of telomere loss
occur as birds age (Monaghan & Ozanne, 2018; Sidorov et al., 2009),
# 1 INTRODUCTION

2 Telomeres are repetitive nucleotide sequences at the terminal regions of eukaryote
3 chromosomes. They serve to protect healthy chromosomes from DNA repair mechanisms that
4 otherwise act on the terminal ends of chromosomes, and organize the replication of DNA during
5 cell division (de Lange 2009). During replication, telomere sequences may be lost, thus
6 shortening the telomere end (Olonikov 1973). Telomerase may replace the lost sequences, thus
7 lengthening it (Chan and Blackburn 2004). Nonetheless, cells with over-shortened telomeres
8 become “senescent” or “self-destruct” (termed apoptosis). When apoptosis occurs, the DNA-
9 encoded information of the cell is removed from the organism (Blackburn 2000). Thus,
10 telomeres may play a role in both cell senescence (or alternatively in cell immortalization due to
11 the activity of telomerase; Tian et al. 2018), accumulation of senescent cells in organs (Campisi
12 2005), and organismal senescence (Blasco 2007; Young 2018). Further, accelerated telomere
13 loss at a given chronological age may indicate decreasing organismal condition, especially during
14 early development, and may underline both physiological stress and a shorter life (viz.,
15 advanced senescence; Haussmann et al. 2003; Bize et al. 2009; Boonekamp et al. 2014; Sudyka
16 et al. 2016; Pepper et al. 2018; Whittemore et al. 2019; Sheldon et al. 2021).

17 Previous studies have suggested that due to the association of rate of telomere loss from
18 chromosomes and organismal senescence, telomere dynamics during life are closely and
19 functionally associated with lifespan among species of different body sizes and pace of life (e.g.,
20 Dantzer and Fletcher 2015; Tricola et al. 2018). These studies primarily focused on birds, and
21 recognized the possible importance of body size and historical patterns (viz., the influence of
22 phylogeny) in explaining a general pattern of relatively slowed loss of telomeres during life in
but they have the disadvantage of including individuals that do not survive to adulthood (Dantzer & Fletcher, 2015). A bias may thus occur between the samples of younger and older birds. Thus, we estimated TROC only from samples of adult birds. A further problem is that some estimates of telomere length measure only DNA sequences of the terminal telomere repeats, whereas other methods include DNA sequences from the body of the chromosome (Remot et al., 2021). We restricted our analyses to those studies that used the former methods and thus produced the best estimates of telomere lengths.

## 2 | MATERIAL AND METHODS

Our earlier study examined associations between telomere variables and aspects of life history for bird species (Criscuolo et al., 2021). Our comparative analysis followed the recommendations of the preferred reporting items for systematics reviews and meta-analysis (PRISMA) statement (Liberati et al., 2009).

In the present study, we focused on 30 species for which adult telomere length, TROC, and life history variables were all recorded. For these species, telomere lengths were estimated in kilobases (kb) using electrophoretic separation of telomere restriction fragments or by quantitative fluorescent in situ hybridization (respectively, TRF and Q-FISH; Remot et al., 2021). All studies used samples of erythrocytes. TROC was measured as the slope of telomere length regressed over age (kb/year) for each of the species (Haussmann et al., 2003; Tricola et al., 2018), but excluding the telomere lengths of hatchling chicks or yearlings (ages of 0-1 year). We followed Dantzer and Fletcher (2015) and Tricola et al. (2018) in using maximum lifespan in nature to typify longevity and mean adult female body mass to typify body size, and log-transformed both variables. Among the 30 species of birds, body-size-independent aspects of longevity (longevity associated with the pace of life) were estimated as the residuals of maximum lifespan regressed on mean female body mass, and these residuals were checked for normality using a Q-Q plot and Shapiro-Wilk test. When multiple sources were available, a mean value of adult telomere length or TROC was used.

We divided lifespan into two statistically independent parts, based on the regression of lifespan onto body size (as estimated by mean adult female body mass for each species). The first variable represented mass-associated aspects of lifespan, as indicated by the predicted values of lifespan from the regression, and termed “mass-predicted lifespan.” The residuals from the analysis were termed “mass-independent lifespan,” and these two measures of lifespan were statistically uncorrelated. The latter variable estimated lifespan at a given body size and may be interpreted as a measure of lifespan on the slow-fast continuum of the pace of life (Dobson & Oli, 2007; Gaillard et al., 1989). Associations of telomere and lifespan variables were examined by comparison of both correlation and by phylogeny-adjusted correlation (after Price, 1997).

A phylogeny was obtained from BirdTree (Figure 1), with 100 phylogenetic trees downloaded from http://www.bird.tree.org (de Magalhaes & Costa, 2009; Jetz et al., 2012) using ape (Paradis & Schliep, 2019), apTreeshape (Orme et al., 2018), and caper R packages (Bortolussi et al., 2020). Branch lengths were estimated using the coalescent method, thus reflecting an estimate of relative divergence times for the phylogeny (Rannala & Yang, 2003). Associations of the bird phylogeny with adult telomere length, TROC, adult female body mass, maximum lifespan, and the residuals of lifespan on body mass were estimated using Hadfield and Nakagawa's (2010) Markov chain Monte Carlo generalized linear mixed model (MCMCglmm; Hadfield, 2010) package in R (R core team, 2020). The MCMCglmm package was also used to produce phylogeny-adjusted estimate of associations of telomere, body mass, and longevity variables.

MCMCglmm was used to produce two types of results: (1) an estimate of the phylogenetic pattern in each of the study variables (viz., $\rho$ = the proportion of variance that could be statistically accounted for by a matrix of the phylogenetic pattern); and (2) degree of correlation of pairs of variables with statistical adjustment to remove the statistical influence of the phylogenetic pattern. Pearson's correlations, unadjusted for phylogeny, were also calculated for comparison with phylogeny-adjusted results. Cohen's (1988) criteria for effect sizes of associations were applied: small $r$ = 0.1, medium $r$ = 0.3, and large $r \ge 0.5$.

## 3 | RESULTS

The regression of maximum lifespan on adult female body mass was highly significant ($R^2$ = 0.59, $F$ = 40.4, $df$ = 1.28, $p$ < .0001). The residuals of this analysis were fairly close to a Gaussian distribution (Shapiro-Wilk statistic = 0.98, $p$ = .84) and were used as an estimate of mass-independent lifespan. The positive association of maximum lifespan and body mass was strong and significant ($r$ = 0.768, $t$ = 6.4, $df$ = 28, $p$ < .0001).

Maximum lifespan had a strong association with the phylogenetic pattern ($\rho$ = 0.805, [credible intervals] $CI_{0.95}$ = 0.284-0.907, $N$ = 30). Body mass (and thus mass-predicted lifespan) also had a strong association with phylogeny ($\rho$ = 0.907, $CI_{0.95}$ = 0.414-0.994, $N$ = 30). Mass-independent lifespan had at best a small association with phylogeny ($\rho$ = 0.073, $CI_{0.95}$ = 0.011-0.495, $N$ = 30). Adult telomere length and TROC had trivial to small associations with the phylogeny (respectively; $\rho$ = 0.073 and 0.062, $CI_{0.95}$ = 0.017-0.497 and 0.018-0.568, $N$ = 30).

Adult telomere length was not significantly correlated with maximum lifespan, mass-predicted lifespan, or mass-independent lifespan (Figure 2a). Once the phylogenetic pattern was taken into account statistically, adult telomere length exhibited trivial to moderate correlations with different estimates of lifespan (adjusted maximum lifespan, $r$ = 0.004, $CI_{0.95}$ = -0.356 - 0.394; adjusted mass-predicted lifespan, $r$ = -0.132, $CI_{0.95}$ = -0.499 - 0.271; adjusted mass-independent lifespan, $r$ = 0.108, $CI_{0.95}$ = -0.232 - 0.768;
23 larger species with syndromes of slower paces of life. These studies applied phylogenetic
24 comparisons to 14 and 19 species of birds (TRF data), respectively, relatively small samples for
25 robust phylogenetic analyses (though Dantzer and Fletcher 2016 presented an analysis that
26 assumed no significant phylogenetic associations). Tricola et al. (2018) suggested that the rate
27 of telomere shortening exhibited a strong historical pattern that may have coevolved with
28 lifespan. Both studies concluded that larger species with relatively slower life histories exhibited
29 reduced rates of telomere shortening.

30 These key previous studies raised a series of questions that might be examined with a
31 larger sample of species of birds. First, how flexible are telomere traits over phylogenetic
32 history? Tricola et al. (2018) found that telomere length was not strongly influenced by the
33 phylogenetic pattern, but telomere rate of change (TROC) was. Alternatively, Criscuolo et al.
34 (2021) found that neither adult telomere length nor TROC showed a strong phylogenetic
35 pattern in a corrected sample size of 52 bird species, a different result that needs to be
36 explained.

37 Second, how do longevity, body size, and the pace of life interact with telomere
dynamics? Dantzer and Fletcher (2015) found that all three variables covaried strongly with
38 TROC, and TROC was lower for the longest-lived bird species. The latter result was confirmed by
39 Tricola et al. (2018) with or without body mass used as a covariate. In a phylogenetic
40 comparative analysis of 9 species of birds and mammals, Le Pepke and Eisenberg (2020)
41 reported a low rate of telomere loss in long-lived species, but a trivial effect of body mass.
42 These studies took a phylogenetic comparative approach where regressions were used to
43 “control” for the phylogenetic patterns underlying variables. Still, none of them quantified how
44
<figure><img src="image_3.png" /></figure>

FIGURE 1 Phylogenetic tree of the 30 bird species for which adult telomere length and telomere length rate of change (TROC) were collected from published papers on avian telomeres using the TRF or Q-FISH methodologies. The consensus phylogenetic tree was obtained from BirdTree.org (100 trees).

all *df* = 27, *p* > .20). It is noteworthy that the adults of large species tended to have shorter telomeres, but at a moderate correlation at best (unadjusted for phylogeny, *r* = -0.249, *t* = 1.4, *N* = 30, *p* = .18).

TROC was significantly positively correlated with lifespan (*r* = 0.497, *t* = 3.0, *df* = 28, *p* < .01), and this pattern was similar but not significant when the phylogenetic pattern was taken into account (adjusted *r* = 0.366, *CI*<sub>0.95</sub> = -0.057 - 0.637, *df* = 27, *p* = .12) (Figure 2b). TROC exhibited a small to medium positive association with mass-predicted lifespan that was not significant (*r* = 0.288, *t* = 1.6, *df* = 28, *p* = .12), and with phylogenetic “adjustment” this association turned slightly negative (adjusted *r* = -0.145, *CI*<sub>0.95</sub> = -0.522 - 0.225, *df* = 27, *p* > .20). TROC and mass-independent lifespan exhibited a moderate but significant positive correlation (*r* = 0.432, *t* = 2.5, *df* = 28, *p* = .02), and the effect size of this association was slightly lower but still significant when the phylogenetic pattern was taken into account statistically (adjusted *r* = 0.327, *CI*<sub>0.95</sub> = 0.037-0.661, *df* = 27, *p* = .04).

4 | DISCUSSION

Our first question concerned the association of life history and
telomere variables with the phylogenetic pattern across 30 spe-
cies of birds. Lifespan and body mass are well known to covary
among bird species and exhibit strong phylogenetic constraint
(e.g., Bennett & Owens, 2002; Criscuolo et al., 2021; Dantzer &
Fletcher, 2015). Less is known of phylogenetic influences on tel-
omere dynamics, but Tricola et al. (2018) suggested little phyloge-
netic influence on telomere length and a significant influence on
TROC. We found the expected fairly strong association of phy-
logenetic pattern and lifespan and mass-predicted lifespan, but a
very weak association of the phylogeny with mass-independent
lifespan (long or short life at a given body size). Telomere length
and the rate of decline in telomere length over time exhibited
weak associations with the phylogeny, contrary to the sugges-
tion of Tricola et al. (2018). Given the fairly strong association
those phylogenetically-controlled patterns of telomere dynamics change with lifespan at a given body size; that is, when influences of body size are statistically controlled (Udroiu 2020).

Third, does telomere length *per se* change with species lifespan, with or without the influences of body size taken into account? Gomes et al. (2011) studied 61 species of mammals, and found that adult telomeres were shorter in the longest-lived species. Both body mass and longevity showed significant negative associations with telomerase and telomere length, respectively, independent of the phylogenetic pattern among the species (Gorbunova and Seluanov 2009). The conclusion of Gomes et al. (2011) was that, for large species to evolve long lifespans, replicative aging occurred (i.e., short telomeres combined with repressed telomerase activity). A result confirmed by a re-analysis of the same dataset recently (Pepke and Eisenberg 2021), with emphasis on an inverse association of telomere length with body mass. These results suggest that short telomeres may have co-evolved in long-lived species as a consequence of body size, underlying the necessity for large species to control for higher risks of cell immortalization by a widespread cellular mechanism (Tian et al. 2018). Interestingly, the phylogeny-adjusted analyses of Tricola et al. (2018) conducted in birds found no significant association of telomere length and longevity, whether or not influences of body mass were included. However, longevity and body mass are known to show strong covariance (Dantzer and Fletcher 2015), and thus may be collinear. The analyses of more bird species by Criscuolo et al. (2021), however, found no significant association of adult telomere length and either body size or the pace of life, with or without inclusion of the phylogenetic pattern in the analyses, though they did not specifically examine longevity and its single-trait association with telomere length.

The purpose of our present study was to examine associations of telomere length and
<figure><img src="image_4.png" /><figcaption>FIGURE 2 (a) Correlations of adult telomere length and three life-history traits of 30 bird species: lifespan, mass-predicted lifespan, and mass-independent lifespan. (b) Correlations of telomere length rate of change (TROC) and the three aspects of longevity. “Lifespan” shows unadjusted correlations, “Mass-Pred Lifespan” shows lifespan values predicted from the regression of lifespan on body size, and “Mass-Ind Lifespan” shows the residuals of the regression of lifespan on body size. Pearson correlations are shown with horizontal center bars and 95% confidence intervals with high and low horizontal bars. Triangles show the phylogeny-adjusted correlation values (none are significantly different from zero at the *p* < .05 level, except for TROC and mass-independent lifespan). The horizontal black dashed lines show zero correlation.</figcaption></figure>

of phylogeny with lifespan and body mass, however, it seemed reasonable to account for the phylogenetic pattern statistically when evaluating associations of lifespan, body mass, and telomere dynamics.

Our second question was whether there was a strong association of lifespan and TROC, as suggested by Dantzer and Fletcher (2015) and Tricola et al. (2018). For this, we considered two aspects of longevity. Large animals live longer, as shown by a large number of studies on life-history traits that scale with body size (e.g., Bennett & Owens, 2002; Dobson & Jouventin, 2007; Gaillard et al., 1989; Read & Harvey, 1989; Roff, 2002; Stearns, 1992). Larger animals take longer to grow to adult size and must allocate considerable resources and effort to maintaining their large number of cells. As such, the first question about longevity is whether it is associated with the overall size of an organism (Dobson, 2007). The second aspect of longevity is associated with the pace of life, along the so-called “slow-fast continuum” (Dobson & Oli, 2007; Gaillard et al., 1989). At a given body size, some species have

greater maximum lifespan than others, and this may be associated with lower reproductive effort, and vice versa for short-lived species. Thus, alternative life-history tactics may be produced among species, at a given body size.

For the first aspect of lifespan that was associated with the size of the species, mass-predicted lifespan had a small association with TROC that was not statistically significant, with or without statistical adjustment for the phylogenetic pattern (note that with adjustment for phylogeny, the sign of this correlation turned negative, Figure 2b). However, our lifespan variable that was independent of body size (viz., mass-independent lifespan) had a moderate positive association with TROC as judged by its effect size, significant with or without statistical adjustment for phylogeny. These results suggest that TROC does not vary strongly with body size per se, but rather has at best a poor association with body size, such that longer-lived species that are somewhat larger exhibited only slightly less telomere loss than somewhat smaller species. However, at a given body size, birds exhibited a stronger pattern of association of relative longevity (i.e., a slow pace-of-life) and TROC. Species with the longest lives for their body mass exhibited the slowest rate of loss of telomeres during life. Thus, the division of lifespan into two parts associated with different aspects of life histories revealed biologically meaningful patterns of varying strengths.

The analyses of Dantzer and Fletcher (2015), Tricola et al. (2018), Udroiu (2020), and Le Pepke and Eisenberg (2020) revealed a general pattern of positive association of longevity and TROC, but without testing for different underlying aspects of longevity. Our results revealed nuances to their conclusions: longevity and TROC showed no consistent pattern of change as body size increased, but rather there was a strong pattern of longevity and TROC increasing together at a given body size. This pattern likely underlies the positive associations of longevity and TROC found by previous studies. Overall, the general agreement of unadjusted and phylogeny-adjusted associations (Figure 2) may suggest that correlations in our results were little influenced by the historical evolutionary pattern reflected by the consensus phylogenetic tree.

Our final question was whether adult telomeres were shorter in the larger and longest-lived species, as suggested by Gomes et al. (2011) and Le Pepke and Eisenberg (2021) for mammals. This latter study suggested that telomere length coevolved with body size, such that large species have short telomeres, and thus facilitated the evolution of long lifespans, notably via the use of cell replication senescence and the reduction of risks of cell immortalization (Risques & Promislow, 2018; Seluanov et al., 2018). On the other hand, Tricola et al. (2018) found a slight but non-significant positive association of telomere length and maximum lifespan among 19 species of birds. While we found that both longevity and body mass followed the phylogenetic pattern fairly closely, telomere dynamics did not. Nonetheless, we found little evidence of larger species having shorter telomeres, with or without statistical adjustment for the influence of the phylogenetic pattern (Figure 2a). In the light of our results, postulating that large birds use replication senescence,
67 TROC on the one hand, and body size and longevity on the other hand in birds. We did this using
68 a restricted sample size of 30 of the bird species (see below for justification) reviewed in the
69 comparative analysis of Criscuolo et al. (2021). We first asked whether any of the variables
70 showed evidence of strong phylogenetic pattern, using the Bayesian meta-analysis approach of
71 Hadfield and Nakagawa (2010). Longevity and body size are closely associated in birds (e.g.,
72 Bennett and Owens 2002; Dantzer and Fletcher 2015; Criscuolo et al. 2021). Larger species
73 reflect many aspects of life histories that covary over evolutionary time, in part because it takes
74 longer to grow and survive to a large body size (Dobson 2007). Further, bird species vary along a
75 "slow-fast continuum" that reflects alternative paces of life that are independent of body size
76 (Gaillard et al. 1989). Thus, we examined variation in longevity that was strongly associated with
77 body size ("mass-predicted lifespan"), and longevity that was statistically independent of body
78 size ("mass-independent lifespan"). The latter reflects changes in lifespan that can be described
79 as varying with the pace of life (Dobson and Oli 2007; Criscuolo et al. 2021). Because differences
80 between phylogenetically-adjusted and unadjusted associations of traits can be biologically
81 informative (Price 1997), we compared both of the aspects of lifespan to telomere length and
82 TROC.

83 We also addressed a further issue with respect to how TROC is measured. First, our
84 sample included estimates of TROC that used mean differences in telomere lengths between
85 chicks and older birds (after Criscuolo et al. 2021). Such estimates have the advantage of
86 including the chick period, when the greatest rates of telomere loss occur as birds age (Sidorov
87 et al. 2009; Monaghan and Ozanne 2018), but they have the disadvantage of including
88 individuals that do not survive to adulthood (Fletcher and Dantzer 2015). A bias may thus occur
as larger mammals do, as a mechanism favoring long lifespan is still an unanswered question. This begs the question of whether at least some bird species have evolved specific anti-aging or anti-cancer mechanisms that are similar to the telomere-related control suggested for long-lived mammalian species that weigh less than a kilogram (Gomes et al., 2011; Tian et al., 2018; but see Seluanov et al., 2018).

Comparative studies like the present one help to point out how aging mechanisms at the cell level may have coevolved with life histories among animal species. So far, as we have seen above, comparative studies have concluded that large body size and long lifespan have evolved with short telomeres and reduced loss of telomeres in mammals, or that longevity and reduced loss of telomeres (but not short telomeres) are matched in birds. This discrepancy might be attributed to the smaller range of sizes in birds, suggesting that if body size and the number of cells are the main constraint to the evolution of long telomeres, this may explain why birds show higher levels of telomere maintenance (e.g., via an enhanced telomerase expression) than mammals and long up-to Mb telomeres (Delany et al., 2000; Monaghan, 2010). Our analysis that controlled for the effects of body size suggested that enhanced telomere maintenance has coevolved with longevity in birds independently of body size, and this differently, even in closely related species. This is, in addition to that of high glycemia and aerobic metabolism, a paradoxical association with avian longevity (Holmes & Harper, 2018), a new aging enigma that requires continued exploration in relation to species' evolutionary histories.

## AUTHOR CONTRIBUTIONS

F. Stephen Dobson: Conceptualization (lead); formal analysis (lead); investigation (lead); writing – original draft (lead); writing – review and editing (equal). **Quentin Schull:** Formal analysis (supporting); writing – review and editing (equal). **François Criscuolo:** Data cura- tion (lead); formal analysis (supporting); writing – review and editing (equal).

## ACKNOWLEDGEMENTS

Our analyses were stimulated by anonymous comments from reviewers of an earlier manuscript (Criscuolo et al., 2021), and we thank them for their thoughtful suggestions. FSD thanks the Institute of Advanced Studies of the University of Strasbourg for their financial support through an USIAS fellowship, and the Région Grand Est and the Eurométropole de Strasbourg for the award of a Gutenberg Excellence Chair. The study was also supported by the Agence Nationale Pour la Recherche, program AGEs, grant number ANR 234061 to FC.

## FUNDING INFORMATION

The present study was supported by a USIAS fellowship and by a Gutenberg Chair awarded to F.S. Dobson. The study was also supported by the Agence Nationale Pour la Recherche, program AGEs, grant number ANR234061 to F. Criscuolo.

## DATA AVAILABILITY STATEMENT

Data file is provided in Appendix S1 and at Dryad [https://doi.org/10.5061/dryad.pnvx0k6rc](https://doi.org/10.5061/dryad.pnvx0k6rc).

## ORCID

F. Stephen Dobson

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

[https://orcid.org/0000-0001-5562-6316](https://orcid.org/0000-0001-5562-6316)

François Criscuolo

<figure><img src="image_6.png" /></figure>

[https://orcid.org/0000-0001-8997-8184](https://orcid.org/0000-0001-8997-8184)

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longevity (longevity associated with the pace of life) were estimated as the residuals of
maximum lifespan regressed on mean female body mass, and these residuals were checked for
normality using a Q-Q plot and Shapiro-Wilk test. When multiple sources were available, a mean
value of adult telomere length or TROC was used. All sources are reported in the online
supplementary information (ESM1).

We divided lifespan into two statistically independent parts, based on the regression of
lifespan onto body size (as estimated by mean adult female body mass for each species). The
first variable represented mass-associated aspects of lifespan, as indicated by the predicted
values of lifespan from the regression, and termed "mass-predicted lifespan." The residuals
from the analysis were termed "mass-independent lifespan," and these two measures of
lifespan were statistically uncorrelated. The latter variable estimated lifespan at a given body
size, and may be interpreted as a measure of lifespan on the slow-fast continuum of the pace of
life (Gaillard et al. 1989; Dobson and Oli 2007). Associations of telomere and lifespan variables
were examined by comparison of both correlation and by phylogeny-adjusted correlation (after
Price 1997).

A phylogeny was obtained from BirdTree (Figure 1), with 100 phylogenetic trees
downloaded from http://www.bird.tree.org (De Magalhaes and Costa 2009; Jetz et al. 2012)
using ape, apTreeshape and caper R packages. Branch lengths were estimated using the
coalescent method, thus reflecting an estimate of relative divergence times for the phylogeny
(Rannala and Yang 2003). Associations of the bird phylogeny with adult telomere length, TROC,
adult female body mass, maximum lifespan, and the residuals of lifespan on body mass were
estimated using Hadfield and Nakagawa's (2010) Markov chain Monte Carlo generalized linear
Young, A. J. (2018). The role of telomeres in the mechanisms and evolution of life-history trade-offs and ageing. *Philosophical Transactions of the Royal Society, B: Biological Sciences*, 373(1741), 20160452.

https://doi.org/10.1098/rstb.2016.0452

## SUPPORTING INFORMATION

Additional supporting information can be found online in the Supporting Information section at the end of this article.

**How to cite this article:** Dobson, F. S., Schull, Q., & Criscuolo, F. (2022). Two aspects of longevity are associated with rates of loss of telomeres in birds. *Ecology and Evolution*, 12, e9364.

https://doi.org/10.1002/ece3.9364
mixed model (MCMCglmm; Hadfield 2010) package in R (R core team 2020). The MCMCglmm package was also used to produce phylogeny-adjusted estimate of associations of telomere, body mass, and longevity variables.

MCMCglmm was used to produce two types of results: 1) an estimate of the phylogenetic pattern in each of the study variables (viz., $\rho$ = the proportion of variance that could be statistically accounted for by a matrix of the phylogenetic pattern); and 2) degree of correlation of pairs of variables with statistical adjustment to remove the statistical influence of the phylogenetic pattern. Pearson's correlations, unadjusted for phylogeny, were also calculated for comparison with phylogeny-adjusted results. Cohen's (1988) criteria for effect sizes of associations were applied: small r = 0.1, medium r = 0.3, and large r $\ge$ 0.5.

## RESULTS

The regression of maximum lifespan on adult female body mass was highly significant ($R^2$ = 0.59, F = 40.4, d.f. = 1,28, P < 0.0001). The residuals of this analysis were fairly close to a Gaussian distribution (Shapiro-Wilk statistic = 0.98, P = 0.84), and were used as an estimate of mass-independent lifespan. The positive association of maximum lifespan and body mass was strong and significant (r = 0.768, t = 6.4, d.f. = 28, P < 0.0001).

Maximum lifespan had a strong association with the phylogenetic pattern ($\rho$ = 0.805, [credible intervals] $CI_{0.95}$ = 0.284 - 0.907, N = 30). Body mass (and thus mass-predicted lifespan) also had a strong association with phylogeny ($\rho$ = 0.907, $CI_{0.95}$ = 0.414 - 0.994, N = 30). Mass-independent lifespan had at best a small association with phylogeny ($\rho$ = 0.073, $CI_{0.95}$ = 0.011 - 0.495, N = 30). Adult telomere length and TROC had trivial to small associations with the
155 phylogeny (respectively; $\rho$ = 0.073 and 0.062, $Cl_{0.95}$ = 0.017 - 0.497 and 0.018 - 0.568, N = 30).

156 Adult telomere length was not significantly correlated with maximum lifespan, mass-157 predicted lifespan, or mass-independent lifespan (Figure 2a). Once the phylogenetic pattern was 158 taken into account statistically, adult telomere length exhibited trivial to moderate correlations 159 with different estimates of lifespan (adjusted maximum lifespan, r = 0.004, $Cl_{0.95}$ = -0.356 - 160 0.394; adjusted mass-predicted lifespan, r = -0.132, $Cl_{0.95}$ = -0.499 - 0.271; adjusted mass-161 independent lifespan, r = 0.108, $Cl_{0.95}$ = -0.232 - 0.768; all d.f. = 27, P > 0.20). It is noteworthy 162 that the adults of large species tended to have shorter telomeres, but at a moderate correlation 163 at best (unadjusted for phylogeny, r = -0.249, t = 1.4, N = 30, P = 0.18).

164 TROC was significantly positively correlated with lifespan (r = 0.497, t = 3.0, d.f. = 28, P < 165 0.01), and this pattern was similar but not significant when the phylogenetic pattern was taken 166 into account (adjusted r = 0.366, $Cl_{0.95}$ = -0.057 - 0.637, d.f. = 27, P = 0.12) (Figure 2b). TROC 167 exhibited a small to medium positive association with mass-predicted lifespan that was not 168 significant (r = 0.288, t = 1.6, d.f. = 28, P = 0.12), and with phylogenetic "adjustment" this 169 association turned slightly negative (adjusted r = -0.145, $Cl_{0.95}$ = -0.522 - 0.225, d.f. = 27, P > 170 0.20). TROC and mass-independent lifespan exhibited a moderate but significant positive 171 correlation (r = 0.432, t = 2.5, d.f. = 28, P = 0.02), and the effect size of this association was 172 slightly lower but still significant when the phylogenetic pattern was taken into account 173 statistically (adjusted r = 0.327, $Cl_{0.95}$ = 0.037 - 0.661, d.f. = 27, P = 0.04).

174

175 **DISCUSSION**
176 Our first question concerned the association of life history and telomere variables with
177 the phylogenetic pattern across 30 species of birds. Lifespan and body mass are well known to
178 covary among bird species and exhibit strong phylogenetic constraint (e.g., Bennett and Owens
179 2002; Dantzer and Fletcher 2015; Criscuolo et al. 2021). Less is known of phylogenetic
180 influences on telomere dynamics, but Tricola et al. (2018) suggested little phylogenetic
181 influence on telomere length and a significant influence on TROC. We found the expected fairly
182 strong association of phylogenetic pattern and lifespan and mass-predicted lifespan, but a very
183 weak association of the phylogeny with mass-independent lifespan (long or short life at a given
184 body size). Telomere length and the rate of decline in telomere length over time exhibited weak
185 associations with the phylogeny, contrary to the suggestion of Tricola et al. (2018). Given the
186 fairly strong association of phylogeny with lifespan and body mass, however, it seemed
187 reasonable to account for the phylogenetic pattern statistically when evaluating associations of
188 lifespan, body mass, and telomere dynamics.

189 Our second question was whether there was a strong association of lifespan and TROC,
190 as suggested by Dantzer and Fletcher (2015) and Tricola et al. (2018). For this, we considered
191 two aspects of longevity. Large animals live longer, as shown by a large number of studies on
192 life-history traits that scale with body size (e.g., Gaillard et al. 1989; Read and Harvey 1989; Roff
193 1992; Stearns 1992; Bennett and Owens 2002; Dobson and Jouventin 2007). Larger animals take
194 longer to grow to adult size and must allocate considerable resources and effort to maintaining
195 their large number of cells. As such, the first question about longevity is whether it is associated
196 with the overall size of an organism (Dobson 2007). The second aspect of longevity is associated
197 with the pace of life, along the so-called "slow-fast continuum" (Gaillard et al. 1989; Dobson and
198 Oli 2007). At a given body size, some species have greater maximum lifespan than others, and
199 this may be associated with lower reproductive effort, and *vice versa* for short-lived species.
200 Thus, alternative life-history tactics may be produced among species, at a given body size.

201 For the first aspect of lifespan, that associated with the size of the species, mass-
202 predicted lifespan had a small association with TROC that was not statistically significant, with
203 or without statistical adjustment for the phylogenetic pattern (note that with adjustment for
204 phylogeny, the sign of this correlation turned negative, Figure 2b). However, our lifespan
205 variable that was independent of body size (viz., mass-independent lifespan) had a moderate
206 positive association with TROC as judged by its effect size, significant with or without statistical
207 adjustment for phylogeny. These results suggest that TROC does not vary strongly with body
208 size *per se*, but rather has at best a poor association with body size, such that longer-lived
209 species that are somewhat larger exhibited only slightly less telomere loss than somewhat
210 smaller species. However, at a given body size, birds exhibited a stronger pattern of association
211 of relative longevity (i.e., a slow pace-of-life) and TROC. Species with the longest lives for their
212 body mass exhibited the slowest rate of loss of telomeres during life. Thus, the division of
213 lifespan into two parts associated with different aspects of life histories revealed biologically
214 meaningful patterns of varying strengths.

215 The analyses of Dantzer and Fletcher (2015), Tricola et al. (2018), Udroiu (2020), and Le
216 Pepke and Eisenberg (2020) revealed a general pattern of positive association of longevity and
217 TROC, but without testing for different underlying aspects of longevity. Our results revealed
218 nuances to their conclusions: longevity and TROC showed no consistent pattern of change as
219 body size increased, but rather there was a strong pattern of longevity and TROC increasing
together at a given body size. This pattern likely underlies the positive associations of longevity and TROC found by previous studies. Overall, the general agreement of unadjusted and phylogeny-adjusted associations (Figure 2) may suggest that correlations in our results were little influenced by the historical evolutionary pattern reflected by the consensus phylogenetic tree.

Our final question was whether adult telomeres were shorter in the larger and longest-lived species, as suggested by Gomes et al. (2011) and Pepke and Eisenberg (2021) for mammals. This latter study suggested that telomere length coevolved with body size, such that large species have short telomeres, and thus facilitated the evolution of long lifespans, notably *via* the use of cell replication senescence and the reduction of risks of cell immortalization (Risques and Promislov 2018; Seluanov et al. 2018). On the other hand, Tricola et al. (2018) found a slight but non-significant positive association of telomere length and maximum lifespan among 19 species of birds. While we found that both longevity and body mass followed the phylogenetic pattern fairly closely, telomere dynamics did not. Nonetheless, we found little evidence of larger species having shorter telomeres, with or without statistical adjustment for the influence of the phylogenetic pattern (Figure 2a). In the light of our results, postulating that large birds use replication senescence, as larger mammals do, as a mechanism favouring long lifespan is still an unanswered question. This begs the question of whether at least some bird species have evolved specific anti-ageing or anti-cancer mechanisms that are similar to the telomere-related control suggested for long-lived mammalian species that weigh less than a kilogram (Gomes et al. 2011; Tian et al. 2018; but see Seluanov et al. 2018).
Comparative studies like the present one help to point out how aging mechanisms at the cell level may have coevolved with life histories among animal species. So far, as we have seen above, comparative studies have concluded that large body size and long lifespan have evolved with short telomeres and reduced loss of telomeres in mammals, or that longevity and reduced loss of telomeres (but not short telomeres) are matched in birds. This discrepancy might be attributed to the smaller range of sizes in birds, suggesting that if body size and the number of cells is the main constraint to the evolution of long telomeres, this may explain why birds show higher levels of telomere maintenance (e.g., via an enhanced telomerase expression) than mammals and long up-to Mb telomeres (Delany et al. 2000; Monaghan 2010). Our analysis that controlled for the effects of body size suggested that enhanced telomere maintenance has coevolved with longevity in birds independently of body size, and this differently, even in closely related species. This is, in addition to that of high glycemia and aerobic metabolism, a paradoxical association with avian longevity (Holmes and Harper 2018), a new aging enigma that requires continued exploration in relation to species' evolutionary histories.

ACKNOWLEDGEMENTS

Our analyses were stimulated by anonymous comments from reviewers of an earlier manuscript (Criscuolo et al. 2021), and we thank them for their thoughtful suggestions. FSD thanks the Institute of Advanced Studies of the University of Strasbourg for their financial support through an USIAS fellowship, and the Région Grand Est and the Eurométropole de Strasbourg for the award of a Gutenberg Excellence Chair. The study was also supported by the Agence Nationale Pour la Recherche, program AGEs, grant number ANR 234061 to FC.
263
264
265 **FUNDING INFORMATION**
266 The present study was supported by a USIAS fellowship and by a Gutenberg Chair awarded to
267 F.S. Dobson.

268
269 **AUTHORS' CONTRIBUTIONS**
270 FC collected the data, FSD wrote the manuscript and analysed the data, and all three authors
271 edited/rewrote the manuscript.

272
273 **DATA ACCESSIBILITY**

274 Data file is provided in ESM1.
275
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407
408
409

410 **FIGURE LEGENDS**

411 Figure 1. Phylogenetic tree of the 30 bird species for which adult telomere length and telomere

412 length rate of change (TROC) were collected from published papers on avian telomeres using
413 the TRF or Q-FISH methodologies. The consensus phylogenetic tree was obtained from

414 BirdTree.org (100 trees).

415

416

417 Figure 2. a) Correlations of adult telomere length and 3 life-history traits of 30 bird species: lifespan, mass-predicted lifespan, and mass-independent lifespan. b) Correlations of telomere length rate of change (TROC) and the 3 aspects of longevity. “Lifespan” shows unadjusted correlations, “Mass-Pred Lifespan” shows lifespan values predicted from the regression of lifespan on body size, and “Mass-Ind Lifespan” shows the residuals of the regression of lifespan on body size. Pearson correlations are shown with horizontal center bars and 95% confidence intervals with high and low horizontal bars. Triangles show the phylogeny-adjusted correlation values (none are significantly different from zero at the P < 0.05 level). The horizontal black dashed lines show zero correlation.

426
427 FIGURES

428 Figure 1. Phylogenetic tree of the 30 bird species for which adult telomere length and telomere

429 length rate of change (TROC) were collected from published papers on avian telomeres using

430 the TRF and q-FISH methodologies. The consensus phylogenetic tree was obtained from

431 BirdTree.org (100 trees).

432
<figure><img src="image_1.png" /></figure>
Figure 2. a) Correlations of adult telomere length (TL) and 3 life-history traits of 30 bird species: lifespan, mass-predicted lifespan, and mass-independent lifespan. b) Correlations of telomere length rate of change (TROC) and the 3 aspects of longevity. “Lifespan” shows unadjusted correlations, “Mass-Pred Lifespan” shows lifespan values predicted from the regression of lifespan on body size, and “Mass-Ind Lifespan” shows the residuals of the regression of lifespan on body size. Pearson correlations are shown with horizontal center bars and 95% confidence intervals with high and low horizontal bars. Triangles show the phylogeny-adjusted correlation values (none are significantly different from zero at the P < 0.05 level). The horizontal black dashed lines show zero correlation.

444
445

446

447

448

449

a)

<figure><img src="image_2.png" /></figure>

b)

<figure><img src="image_3.png" /></figure>