01 - Introduction

Fluctuations in key climatic variables like environmental temperature can affect the energy balance of vertebrate populations [1,2] 1. Angilletta MJ, Cooper BS, Schuler MS, Boyles JG. 2010 The evolution of thermal physiology in endotherms. Front. Biosci. 2, 861-881.
2. Buckley LB, Hurlbert AH, Jetz W. 2012 Broad-scale ecological implications of ectothermy and endothermy in changing environments. Global Ecol. Biogeogr. 21, 873-885. (doi:10.1111/j.1466-8238.2011.00737.x) . Individuals from wild populations respond by displaying phenotypic plasticity (or ‘flexibility’), to reversibly and repeatedly adjust their expression of thermoregulatory, metabolic, and behavioural traits [3,4]3. Kingsolver JG, Huey RB. 1998 Evolutionary analyses of morphological and physiological plasticity in thermally variable environments. Am. Zool. 38, 545-560. (doi:10.1093/icb/38.3.545)
4. Tattersall GJ, Sinclair BJ, Withers PC, Fields PA, Seebacher F, Cooper CE, Maloney SK. 2012 Coping with thermal challenges: physiological adaptations to environmental temperatures. Comp. Physiol. 2, 2151-2202. (doi:10.1002/cphy.c110055) . Glucocorticoids, a conserved set of vertebrate steroid hormones, transmit information about environmental variation like temperature fluctuations by rapidly changing their concentrations in the blood [5-7] 5. Wingfield JC, Ramenofsky M. 2011 Hormone-behavior interrelationships of birds in response to weather. In Advances in the study of behavior, 43 (eds Brockmann HJ, Roper TJ, Naguib M, Mitani JC, Leigh WS), pp. 93-188. New York, NY: Academic Press. Google Scholar
6. de Bruijn R, Romero LM. 2018 The role of glucocorticoids in the vertebrate response to weather. Gen. Comp. Endocrinol. 269, 11-32. (doi:10.1016/j.ygcen.2018.07.007)
7. Jessop TS, Lane ML, Teasdale L, Stuart-Fox D, Wilson RS, Careau V, Moore IT. 2016 Multiscale evaluation of thermal dependence in the glucocorticoid response of vertebrates. Am. Nat. 188, 342-356. (doi:10.1086/687588) . Glucocorticoid receptors are distributed in various parts of the brain and body [8–10] 8. Lattin CR, Waldron-Francis K, Richardson JW, de Bruijn R, Bauer CM, Breuner CW, Romero L. 2012 Pharmacological characterization of intracellular glucocorticoid receptors in nine tissues from house sparrow (Passer domesticus). Gen. Comp. Endocrinol. 179, 214-220. (doi:10.1016/j.ygcen.2012.08.007)
9. under JW. 2005 Mineralocorticoid receptors: distribution and activation. Heart Fail. Rev. 10, 15-22. (doi:10.1007/s10741-005-2344-2)
Senft RA, Meddle SL, Baugh AT. 2016 Distribution and abundance of glucocorticoid and mineralocorticoid receptors throughout the brain of the great tit (Parus major). PLoS ONE 11, e0148516. (doi:10.1371/journal.pone.0148516) , enabling these hormones to function as higher-order regulators to pleiotropically promote phenotypic plasticity [11–13] 11. Cox RM, McGlothlin JW, Bonier F. 2016 Hormones as mediators of phenotypic and genetic integration: an evolutionary genetics approach. Integr. Comp. Biol. 56, 126-137. (doi:10.1093/icb/icw033)
12. Wingfield JC, Maney DL, Breuner CW, Jacobs JD, Lynn S, Ramenofsky M, Richardson R. 1998 Ecological bases of hormone—behavior interactions: the ‘emergency life history stage'. Am. Zool. 38, 191-206. (doi:10.1093/icb/38.1.191)
13. Hau M, Casagrande S, Ouyang JQ, Baugh AT. 2016 Glucocorticoid-mediated phenotypes in vertebrates: multilevel variation and evolution. In Advances in the study of behavior, 48 (eds Naguib M, Mitani JC, Simmons LW, Barrett L, Healy S, Zuk M), pp. 41-115. New York, NY: Academic Press. . For these reasons, the plasticity of glucocorticoid responses has been suggested as one of the mechanisms underlying within- and among-individual differences in phenotypic plasticity [13–16] 13. Hau M, Casagrande S, Ouyang JQ, Baugh AT. 2016 Glucocorticoid-mediated phenotypes in vertebrates: multilevel variation and evolution. In Advances in the study of behavior, 48 (eds Naguib M, Mitani JC, Simmons LW, Barrett L, Healy S, Zuk M), pp. 41-115. New York, NY: Academic Press.
14. Taff CC, Vitousek MN. 2016 Endocrine flexibility: optimizing phenotypes in a dynamic world? Trends Ecol. Evol. 31, 476-488. (doi:10.1016/j.tree.2016.03.005)
15. Wada H, Sewall KB. 2014 Introduction to the symposium—uniting evolutionary and physiological approaches to understanding phenotypic plasticity. Integr. Comp. Biol. 54, 774-782. (doi:10.1093/icb/icu097)
16. Guindre-Parker S. 2020 Individual variation in glucocorticoid plasticity: considerations and future directions. Integr. Comp. Biol. 60, 79-88. (doi:10.1093/icb/icaa003) .

Individuals can differ in how strongly they respond to variation in environmental temperature in plastic changes of metabolic rate [17,18] 17. Kar F, Nakagawa S, Friesen CR, Noble DWA. 2021 Individual variation in thermal plasticity and its impact on mass-scaling. Oikos 130, 1131-1142. (doi:10.1111/oik.08122)
18. Careau V, Gifford ME, Biro PA. 2014 Individual (co)variation in thermal reaction norms of standard and maximal metabolic rates in wild-caught slimy salamanders. Funct. Ecol. 28, 1175-1186. (doi:10.1111/1365-2435.12259) or foraging activity [19]Hall LE, Chalfoun AD. 2019 Behavioural plasticity modulates temperature-related constraints on foraging time for a montane mammal. J. Anim. Ecol. 88, 363-375. (doi:10.1111/1365-2656.12925) . Quantifying such individual differences in the glucocorticoid response in wild vertebrate populations is crucial for evaluating the mechanisms shaping within- and among-individual differences in phenotypic plasticity and to elucidate their evolutionary potential (e.g. [13,14–16,20,21] 13. Hau M, Casagrande S, Ouyang JQ, Baugh AT. 2016 Glucocorticoid-mediated phenotypes in vertebrates: multilevel variation and evolution. In Advances in the study of behavior, 48 (eds Naguib M, Mitani JC, Simmons LW, Barrett L, Healy S, Zuk M), pp. 41-115. New York, NY: Academic Press.
14. Taff CC, Vitousek MN. 2016 Endocrine flexibility: optimizing phenotypes in a dynamic world? Trends Ecol. Evol. 31, 476-488. (doi:10.1016/j.tree.2016.03.005)
15. Wada H, Sewall KB. 2014 Introduction to the symposium—uniting evolutionary and physiological approaches to understanding phenotypic plasticity. Integr. Comp. Biol. 54, 774-782. (doi:10.1093/icb/icu097)
16. Guindre-Parker S. 2020 Individual variation in glucocorticoid plasticity: considerations and future directions. Integr. Comp. Biol. 60, 79-88. (doi:10.1093/icb/icaa003)
20. Angelier F, Wingfield JC. 2013 Importance of the glucocorticoid stress response in a changing world: theory, hypotheses and perspectives. Gen. Comp. Endocrinol. 190, 118-128. (doi:10.1016/j.ygcen.2013.05.022)
21. Malkoc K, Mentesana L, Casagrande S, Hau M. 2021 Quantifying glucocorticoid plasticity using reaction norm approaches: there still is so much to discover! Integr. Comp. Biol. 62, 58-70. ). Alterations in environmental temperatures across the globe are one of the most prominent manifestations of the ongoing climate change [22] 22. IPCC. 2021 Climate change 2021: the physical science basis. In Contribution of working group I to the sixth assessment report of the intergovernmental panel on climate change (Masson-Delmotte eds V et al.) Cambridge, UK: Cambridge University Press.. The predicted increases in yearly average temperatures and in the occurrence of temperature extremes will probably alter the selection pressures that wild populations are exposed to. It is expected that populations which exhibit among-individual variation in plastic responses to environmental temperature are better equipped to withstand climate change and if the standing phenotypic variation has a heritable component, may evolutionarily adapt to these changes (e.g. [23,24] 23. Ruuskanen S, Hsu B-Y, Nord A. 2021 Endocrinology of thermoregulation in birds in a changing climate. Mol. Cell. Endocrinol. 519, 111088. (doi:10.1016/j.mce.2020.111088)
24. Hoffmann AA, Sgro CM. 2011 Climate change and evolutionary adaptation. Nature 470, 479-485. (doi:10.1038/nature09670) ).

To our knowledge, it is presently unknown whether individuals from free-living vertebrate populations differ in their glucocorticoid plasticity when experiencing natural fluctuations in environmental temperature. Addressing this question requires a reaction norm approach, which in its simplest form describes a linear relationship between an individual's repeatedly measured phenotype (e.g. glucocorticoid concentrations) and an environmental or internal gradient (e.g. environmental temperature) [25,26] 25. Nussey DH, Wilson AJ, Brommer JE. 2007 The evolutionary ecology of individual phenotypic plasticity in wild populations. J. Evol. Biol. 20, 831-844. (doi:10.1111/j.1420-9101.2007.01300.x)
26. Dingemanse NJ, Kazem AJN, Réale D, Wright J. 2010 Behavioural reaction norms: animal personality meets individual plasticity. Trends Ecol. Evol. 25, 81–89. . Using linear mixed models, phenotypic plasticity can be decomposed into the elevation (the average value of a trait in the average environment), the slope of the response to the environment (the change in this trait across the gradient, i.e. its plasticity), and their covariance (i.e. whether links exist between these two reaction norm components that may constrain their variation) [25,26] 25. Nussey DH, Wilson AJ, Brommer JE. 2007 The evolutionary ecology of individual phenotypic plasticity in wild populations. J. Evol. Biol. 20, 831-844. (doi:10.1111/j.1420-9101.2007.01300.x)
26. Dingemanse NJ, Kazem AJN, Réale D, Wright J. 2010 Behavioural reaction norms: animal personality meets individual plasticity. Trends Ecol. Evol. 25, 81–89. . These hierarchical analyses allow to separately quantify glucocorticoid elevation (average concentrations) and slope (plastic changes) for each individual (within-individual level), while also estimating whether differences among individuals from this population exist in average concentrations and/or their plasticity to a gradient in environmental temperature.

Reaction norm analyses can be used to analyse glucocorticoid variation separately for baseline and stress-induced concentrations, and to test whether they covary. While glucocorticoid concentrations in individuals going about their lives hardly ever are ‘basal’ and at the lowest possible level, samples taken within 2–3 min of capture, the time lag of the hypothalamo-pituitary-adrenal (HPA) axis to respond to a disturbance, are usually referred to as ‘baseline’ and considered to reflect normal daily activities [27–29] 27. Wingfield JC, Smith JP, Farner DS. 1982 Endocrine responses of white-crowned sparrows to environmental stress. Condor 84, 399-409. (doi:10.2307/1367443)
28. Romero LM, Reed JM. 2005 Collecting baseline corticosterone samples in the field: is under 3 min good enough? Comp. Biochem. Physiol. A 140, 73-79. (doi:10.1016/j.cbpb.2004.11.004)
29. Small TW, Bebus SE, Bridge ES, Elderbrock EK, Ferguson SM, Jones BC, Schoech SJ. 2017 Stress-responsiveness influences baseline glucocorticoid levels: revisiting the under 3 min sampling rule. Gen. Comp. Endocrinol. 247, 152-165. (doi:10.1016/j.ygcen.2017.01.028) . By contrast, ‘stress-induced’ concentrations indicate samples taken 30–60 min after a major disturbance that activated the HPA axis and resulted in a major increase in blood glucocorticoid concentrations [30]30. Sapolsky RM, Romero LM, Munck AU. 2000 How do glucocorticoids influence stress-responses? Integrating permissive, suppressive, stimulatory, and preparative actions. Endocr Rev. 21, 55-89. (doi:10.1210/edrv.21.1.0389) . Separate analyses at these distinct concentrations are justified because glucocorticoids activate different genomic receptors and can exert divergent phenotypic effects [30–33] 30. Sapolsky RM, Romero LM, Munck AU. 2000 How do glucocorticoids influence stress-responses? Integrating permissive, suppressive, stimulatory, and preparative actions. Endocr Rev. 21, 55-89. (doi:10.1210/edrv.21.1.0389)
31. Landys MM, Ramenofsky M, Wingfield JC. 2006 Actions of glucocorticoids at a seasonal baseline as compared to stress-related levels in the regulation of periodic life processes. Gen. Comp. Endocrinol. 148, 132-149. (doi:10.1016/j.ygcen.2006.02.013)
32.Koolhaas JM et al.. 2011 Stress revisited: a critical evaluation of the stress concept. Neurosci. Biobehav. Rev. 35, 1291-1301. (doi:10.1016/j.neubiorev.2011.02.003)
33. Romero LM. 2004 Physiological stress in ecology: lessons from biomedical research. Trends Ecol. Evol. 19, 249-255. (doi:10.1016/j.tree.2004.03.008) . Comparative work across vertebrate taxa also suggests that baseline and stress-induced corticosterone concentrations are shaped by different selection pressures [34]34. Vitousek MN et al.. 2019 Macroevolutionary patterning in glucocorticoids suggests different selective pressures shape baseline and stress-induced levels. Am. Nat. 193, 866-880. . At low baseline levels, circulating glucocorticoid concentrations exert their actions by binding to the high-affinity mineralocorticoid receptor to promote glucose availability to tissues, stimulating appetite, and regulating protein and lipid stores [30,31] 30. Sapolsky RM, Romero LM, Munck AU. 2000 How do glucocorticoids influence stress-responses? Integrating permissive, suppressive, stimulatory, and preparative actions. Endocr Rev. 21, 55-89. (doi:10.1210/edrv.21.1.0389)
31. Landys MM, Ramenofsky M, Wingfield JC. 2006 Actions of glucocorticoids at a seasonal baseline as compared to stress-related levels in the regulation of periodic life processes. Gen. Comp. Endocrinol. 148, 132-149. (doi:10.1016/j.ygcen.2006.02.013) . This supports predictable variations in energy demands in response to changes in weather, life-history stage, and time of day, thereby promoting plastic adjustments in behavioural and physiological traits like foraging, offspring provisioning, or metabolic rate [31] 31. Landys MM, Ramenofsky M, Wingfield JC. 2006 Actions of glucocorticoids at a seasonal baseline as compared to stress-related levels in the regulation of periodic life processes. Gen. Comp. Endocrinol. 148, 132-149. (doi:10.1016/j.ygcen.2006.02.013) . Whenever conditions change unpredictably, inducing major energetic or psychological challenges, glucocorticoids increase within a few minutes to reach high stress-induced concentrations, which then also occupy the low-affinity glucocorticoid receptor [30,32,35] 30. Sapolsky RM, Romero LM, Munck AU. 2000 How do glucocorticoids influence stress-responses? Integrating permissive, suppressive, stimulatory, and preparative actions. Endocr Rev. 21, 55-89. (doi:10.1210/edrv.21.1.0389)
32. Koolhaas JM et al.. 2011 Stress revisited: a critical evaluation of the stress concept. Neurosci. Biobehav. Rev. 35, 1291-1301. (doi:10.1016/j.neubiorev.2011.02.003)
Romero LM, Dickens MJ, Cyr NE. 2009 The reactive scope model – a new model integrating homeostasis, allostasis, and stress. Horm. Behav. 55, 375-389. (doi:10.1016/j.yhbeh.2008.12.009) . This mechanism shifts an individual into an ‘emergency state’, which can involve mobilization of its energy stores, re-allocation of energetic resources to processes that facilitate coping with and recovering from the immediate challenge, and the inhibition of reproductive behaviours that are not essential for surviving the challenge [12,30] 12. Wingfield JC, Maney DL, Breuner CW, Jacobs JD, Lynn S, Ramenofsky M, Richardson R. 1998 Ecological bases of hormone—behavior interactions: the ‘emergency life history stage'. Am. Zool. 38, 191-206. (doi:10.1093/icb/38.1.191)
30. Sapolsky RM, Romero LM, Munck AU. 2000 How do glucocorticoids influence stress-responses? Integrating permissive, suppressive, stimulatory, and preparative actions. Endocr Rev. 21, 55-89. (doi:10.1210/edrv.21.1.0389) .

Recent work suggests that individual differences in glucocorticoid reaction norm elevation and slope exist in wild vertebrate populations (summaries: [14,16,21] 14. Taff CC, Vitousek MN. 2016 Endocrine flexibility: optimizing phenotypes in a dynamic world? Trends Ecol. Evol. 31, 476-488. (doi:10.1016/j.tree.2016.03.005)
16. Guindre-Parker S. 2020 Individual variation in glucocorticoid plasticity: considerations and future directions. Integr. Comp. Biol. 60, 79-88. (doi:10.1093/icb/icaa003)
21. Malkoc K, Mentesana L, Casagrande S, Hau M. 2021 Quantifying glucocorticoid plasticity using reaction norm approaches: there still is so much to discover! Integr. Comp. Biol. 62, 58-70. ). However, some of these studies determined glucocorticoid metabolites from excrements (urine: [36]36. Sonnweber R et al.. 2018 Circadian rhythms of urinary cortisol levels vary between individuals in wild male chimpanzees: a reaction norm approach. Front. Ecol. Evol. 6, 85. (doi:10.3389/fevo.2018.00085) , faeces: [37]37. Guindre-Parker S, McAdam AG, van Kesteren F, Palme R, Boonstra R, Boutin S, Lane JE, Dantzer B. 2019 Individual variation in phenotypic plasticity of the stress axis. Biol. Lett. 15, 20190260. (doi:10.1098/rsbl.2019.0260) ), which poses several biological and methodological challenges that make the interpretation of results and comparisons across species and studies difficult (reviews: [38,39] 38. Gormally BMG, Romero LM. 2020 What are you actually measuring? A review of techniques that integrate the stress response on distinct time-scales. Funct. Ecol. 34, 2030-2044. (doi:10.1111/1365-2435.13648)
39. Dantzer B, Fletcher QE, Boonstra R, Sheriff MJ. 2014 Measures of physiological stress: a transparent or opaque window into the status, management and conservation of species? Conserv. Physiol. 2, cou023. (doi:10.1093/conphys/cou023) ). So far, studies in the wild that measured blood glucocorticoid concentrations focused on aspects of the ‘stress-response’, i.e. the increase from baseline to stress-induced concentrations [40,41] 40. Lendvai ÁZ, Giraudeau M, Bókony V, Angelier F, Chastel O. 2015 Within-individual plasticity explains age-related decrease in stress response in a short-lived bird. Biol. Lett. 11, 20150272. (doi:10.1098/rsbl.2015.0272)
41. Grant AR, Baldan D, Kimball MG, Malisch JL, Ouyang JQ. 2020 Across time and space: hormonal variation across temporal and spatial scales in relation to nesting success. Gen. Comp. Endocrinol. 292, 113462. (doi:10.1016/j.ygcen.2020.113462) . Some studies in captivity documented individual variation in circulating baseline glucocorticoid plasticity (in birds; [42,43] 42. Baldan D, Negash M, Ouyang JQ. 2021 Are individuals consistent? Endocrine reaction norms under different ecological challenges. J. Exp. Biol. 224, jeb.240499. (doi:10.1242/jeb.240499)
43. Lendvai ÁZ, Ouyang JQ, Schoenle LA, Fasanello V, Haussmann MF, Bonier F, Moore IT. 2014 Experimental food restriction reveals individual differences in corticosterone reaction norms with no oxidative costs. PLoS ONE 9, e110564. (doi:10.1371/journal.pone.0110564) ), while others found individual differences only in glucocorticoid elevation (in water-borne hormone samples of fishes; [44,45] 44. Houslay TM, Earley RL, Young AJ, Wilson AJ. 2019 Habituation and individual variation in the endocrine stress response in the Trinidadian guppy (Poecilia reticulata). Gen. Comp. Endocrinol. 270, 113-122. (doi:10.1016/j.ygcen.2018.10.013)
45. Fürtbauer I, Pond A, Heistermann M, King AJ. 2015 Personality, plasticity and predation: linking endocrine and behavioural reaction norms in stickleback fish. Funct. Ecol. 29, 931-940. (doi:10.1111/1365-2435.12400) ). Findings in captivity can be of limited relevance for natural situations because individuals may be chronically stressed and/or lack natural cues, which can bias glucocorticoid concentrations (e.g. [46]46. Calisi RM, Bentley GE. 2009 Lab and field experiments: are they the same animal? Horm. Behav. 56, 1-10. (doi:10.1016/j.yhbeh.2009.02.010) ).

We therefore conducted a 5-year study in a population of free-living great tits (Parus major) in which we applied a reaction norm approach to determine within- and among-individual variation in corticosterone plasticity (the main avian glucocorticoid) along a natural gradient in environmental temperature. We repeatedly sampled adults of both sexes for circulating concentrations of baseline and stress-induced corticosterone using a standardized capture-restraint protocol [27]27. Wingfield JC, Smith JP, Farner DS. 1982 Endocrine responses of white-crowned sparrows to environmental stress. Condor 84, 399-409. (doi:10.2307/1367443) during a specific reproductive stage, the peak nestling provisioning phase. Repeated sampling mostly occurred across years, since in our population great tits infrequently initiate a second clutch. Using a series of general linear-mixed effect models (GLMMs), we tested the following predictions:

(i) baseline and stress-induced corticosterone concentrations increase with lower environmental temperature at the population level, as is commonly found in endotherms (summaries: [5,6,7,23] 5. Wingfield JC, Ramenofsky M. 2011 Hormone-behavior interrelationships of birds in response to weather. In Advances in the study of behavior, 43 (eds Brockmann HJ, Roper TJ, Naguib M, Mitani JC, Leigh WS), pp. 93-188. New York, NY: Academic Press.
6. de Bruijn R, Romero LM. 2018 The role of glucocorticoids in the vertebrate response to weather. Gen. Comp. Endocrinol. 269, 11-32. (doi:10.1016/j.ygcen.2018.07.007)
7. Jessop TS, Lane ML, Teasdale L, Stuart-Fox D, Wilson RS, Careau V, Moore IT. 2016 Multiscale evaluation of thermal dependence in the glucocorticoid response of vertebrates. Am. Nat. 188, 342-356. (doi:10.1086/687588)
23. Ruuskanen S, Hsu B-Y, Nord A. 2021 Endocrinology of thermoregulation in birds in a changing climate. Mol. Cell. Endocrinol. 519, 111088. (doi:10.1016/j.mce.2020.111088) ). Baseline and stress-induced corticosterone concentrations are predicted to be better associated with short-term (temperature at capture) than with longer-term temperature measurements (temperature averages on capture day or on days preceding capture) because small endotherms should track short-term temperature changes as they usually carry little energy reserves;

(ii) average (elevation of the reaction norm) baseline and stress-induced corticosterone concentrations differ among individuals (e.g. meta-analyses: [47,48] 47. Schoenemann KL, Bonier F. 2018 Repeatability of glucocorticoid hormones in vertebrates: a meta-analysis. PeerJ. 6, e4398. (doi:10.7717/peerj.4398)
48. Taff CC, Schoenle LA, Vitousek MN. 2018 The repeatability of glucocorticoids: a review and meta-analysis. Gen. Comp. Endocrinol. 260, 136-145. (doi:10.1016/j.ygcen.2018.01.011) );

(iii) within individuals, corticosterone concentrations change plastically and increase with lower environmental temperatures, especially when these are below the thermoneutral zone (the range of environmental temperatures at which individuals do not expend energy on thermoregulation; approximately 15–30°C in great tits [49,50] 49. Broggi J, Hohtola E, Orell M, Nilsson J-Å, Benkman C. 2005 Local adaptation to winter conditions in a passerine spreading north: a common-garden approach. Evolution 59, 1600-1603. (doi:10.1111/j.0014-3820.2005.tb01810.x)
Malkoc K, Casagrande S, Hau M. 2021 Inferring whole-organism metabolic rate from red blood cells in birds. Front. Physiol. 12, 1066. (doi:10.3389/fphys.2021.691633) );

(iv) the reaction norm slope (plasticity) in response to environmental temperature differs among individuals, with some individuals showing greater corticosterone plasticity (steeper slope) than others (e.g. [42] 42. Baldan D, Negash M, Ouyang JQ. 2021 Are individuals consistent? Endocrine reaction norms under different ecological challenges. J. Exp. Biol. 224, jeb.240499. (doi:10.1242/jeb.240499) );

(v) elevation and slope covary among individuals as in previous work [44]44. Houslay TM, Earley RL, Young AJ, Wilson AJ. 2019 Habituation and individual variation in the endocrine stress response in the Trinidadian guppy (Poecilia reticulata). Gen. Comp. Endocrinol. 270, 113-122. (doi:10.1016/j.ygcen.2018.10.013) . This has rarely been tested (summarized by: [16]16. Guindre-Parker S. 2020 Individual variation in glucocorticoid plasticity: considerations and future directions. Integr. Comp. Biol. 60, 79-88. (doi:10.1093/icb/icaa003) ); and

(vi) baseline and stress-induced corticosterone concentrations show positive covariation at the population and within-individual levels, but not at the among-individual level (e.g. [51]51. Baugh AT, van Oers K, Dingemanse NJ, Hau M. 2014 Baseline and stress-induced glucocorticoid concentrations are not repeatable but covary within individual great tits (Parus major). Gen. Comp. Endocrinol. 208, 154-163. (doi:10.1016/j.ygcen.2014.08.014) ). Population-level correlations, i.e. higher baseline concentrations at capture being associated with higher stress-induced concentrations, could arise from within-individual processes like a concerted change in both traits because an individual's overall state is altered by environmental temperature. A non-exclusive alternative are among-individual processes, i.e. when an individual consistently displays higher concentrations in both corticosterone traits compared to conspecifics, owing to genetic, maternal, or environmental factors (e.g. [51]51. Baugh AT, van Oers K, Dingemanse NJ, Hau M. 2014 Baseline and stress-induced glucocorticoid concentrations are not repeatable but covary within individual great tits (Parus major). Gen. Comp. Endocrinol. 208, 154-163. (doi:10.1016/j.ygcen.2014.08.014) ).

02 - Methods

(a) Field Work

Individually marked adult great tits were studied in a free-living nestbox population in the Ettenhofer Holz, Upper Bavaria, Germany from early May to early July of 2015 through to 2019. The birds were captured when entering the nestbox to feed their 8-day-old nestlings (hatching = day 0; further details in the electronic supplementary material).

(b) Statistical analyses

The dataset for baseline corticosterone contained 389 measurements from 240 individuals. Among them, 140 individuals had one measurement, 68 individuals had two, 19 had three, nine had four and four individuals had five. For stress-induced corticosterone, the dataset contained 376 measurements from 237 individuals. Among them, 144 individuals had one measurement, 62 individuals had two, 19 had three, nine had four and three individuals had five. Most repeated measures were obtained in different years (further details in the electronic supplementary material). We included data from all individuals in all our analyses, i.e. also those that were sampled only once. This methodology improves power while yielding similar variance estimates with narrower confidence intervals [52]Martin JGA, Nussey DH, Wilson AJ, Réale D. 2011 Measuring individual differences in reaction norms in field and experimental studies: a power analysis of random regression models. Methods Ecol. Evol. 2, 362-374. (doi:10.1111/j.2041-210X.2010.00084.x) . For all models, we visually inspected the residuals of the data and found them to qualitatively follow standard criteria for residual normality.

03 - Results

(a) Population-level responses to temperature, and individual differences in average concentrations

Variation in baseline and stress-induced corticosterone was best explained by environmental temperature at the moment of capture (DIC model comparisons of model 0, second-best model with maximum temperature at day of capture: ΔAIC > 4 for baseline and stress-induced corticosterone, respectively; electronic supplementary material, tables S1 and S2). Baseline and stress-induced corticosterone were both negatively related to temperature at capture (model 1, table 2, figure 1), i.e. baseline and stress-induced corticosterone levels were higher when temperatures at capture were lower (range of variation: approx. 4–27°C, electronic supplementary material, figures S1 and S2). This significant effect of temperature at capture was somewhat stronger for stress-induced corticosterone than for baseline corticosterone (table 2, figure 1), indicating that stress-induced corticosterone might be even more conditioned by local temperatures than baseline concentrations. Furthermore, as expected from ample previous work including on great tits [27–29,59] 27. Wingfield JC, Smith JP, Farner DS. 1982 Endocrine responses of white-crowned sparrows to environmental stress. Condor 84, 399-409. (doi:10.2307/1367443)
28. Romero LM, Reed JM. 2005 Collecting baseline corticosterone samples in the field: is under 3 min good enough? Comp. Biochem. Physiol. A 140, 73-79. (doi:10.1016/j.cbpb.2004.11.004)
29. Small TW, Bebus SE, Bridge ES, Elderbrock EK, Ferguson SM, Jones BC, Schoech SJ. 2017 Stress-responsiveness influences baseline glucocorticoid levels: revisiting the under 3 min sampling rule. Gen. Comp. Endocrinol. 247, 152-165. (doi:10.1016/j.ygcen.2017.01.028)
59. Baugh AT, van Oers K, Naguib M, Hau M. 2013 Initial reactivity and magnitude of the acute stress response associated with personality in wild great tits (Parus major). Gen. Comp. Endocrinol. 189, 96-104. (doi:10.1016/j.ygcen.2013.04.030) , baseline, but not stress-induced corticosterone, was influenced by the time needed to complete a blood sample, with longer sampling times being associated with higher concentrations of baseline corticosterone. We also found large year differences, and, to a lower extent, sex differences for both hormonal traits (model 1, table 2). Both hormonal traits were repeatable: individual differences in average trait expression explained 17% of the total population variance in baseline corticosterone, and 26% in stress-induced corticosterone (table 2).

Table 2. Sources of phenotypic variation in baseline and stress-induced corticosterone concentrations (model 1). (Both variables were divided by 1000 and modelled following a Gaussian error distribution. Estimates of fixed (β) and random (σ2) parameters are shown as posterior modes with 95% credible intervals (CI). Repeatability, conditional to the variance explained by the fixed effects, was estimated as the proportion of the total phenotypic variance explained by individual variance.)

fixed effects	baseline corticosterone		stress-induced corticosterone
	β	95% CI	β	95% CI
intercept	6.83	[5.593, 8.16]	24.03	[21.224, 26.999]
sex [female]	0.69	[0.025, 1.226]	2.43	[0.194, 4.912]
year [2016]	−1.67	[−3.055, −0.364]	−6.43	[−10.053, −2.803]
year [2017]	−1.91	[−3.263, −0.548]	−1.33	[−4.588, 2.077]
year [2018]	−1.24	[−2.632, 0.302]	7.35	[3.26, 11.122]
year [2019]	−3.38	[−4.78, −2.097]	−0.74	[−3.805, 2.544]
bleeding time (scaled)	1.00	[0.672, 1.296]	0.75	[−0.411, 1.902]
temperature at capture (scaled)	−0.62	[−0.856, −0.353]	−2.39	[−3.545, −1.284]
random effects	σ2	95%CI	σ2	95%CI
individual
intercept	1.90	[0.804, 3.061]	41.87	[22.058, 60.442]
linear slope	0.29	[0.001, 0.658]	3.60	[0.009, 10.882]
intercept–slope covariance	−0.47	[−0.957, 0.012]	−2.88	[−9.884, 2.937]
intercept–slope correlation	−0.65	[−0.987, −0.207]	−0.28	[−0.895, 0.352]
residual 1	30.88	[20.679, 41.038]	111.31	[66.323, 159.672]
residual 2	6.21	[3.768, 8.923]	102.74	[65.293, 141.702]
residual 3	5.96	[3.96, 8.473]	81.00	[45.969, 112.406]
residual 4	6.42	[3.671, 9.286]	102.66	[58.087, 153.517]
residual 5	1.15	[0.277, 2.318]	15.26	[0.281, 38.185]

Figure 1. Predictions from two random regression models of baseline (a) and stress-induced (b) corticosterone concentrations in response to environmental temperature at capture (mean-centred and variance standardized). Each yellow solid line represents a single individual, the dashed black line represents the population-level response to temperature, and grey dots represent the raw phenotypic data (colours are provided in the online version of the figure).

04 - Discussion

We applied a reaction norm approach to quantify variation in circulating glucocorticoid concentrations in free-living great tits sampled repeatedly across different breeding events over five years. As predicted, individuals from our study population plastically changed circulating baseline and stress-induced corticosterone concentrations in response to the environmental temperature they experienced at capture in each year during the peak nestling provisioning phase (figure 1; electronic supplementary material, figure S2). Importantly, our analyses revealed that individuals differed in both reaction norm components for baseline and stress-induced corticosterone concentrations: in average concentrations (i.e. reaction norm elevations) and in their plasticity (reaction norm slopes), with some individuals changing corticosterone concentrations more strongly in response to environmental temperature than others (figure 1; electronic supplementary material, figure S2). These findings provide unique evidence that plastic endocrine responses to a key ecological factor vary among and within individual free-living great tits. Thus, if individual variation in glucocorticoid plasticity was heritable (e.g. [60,61] 60. Stedman JM, Hallinger KK, Winkler DW, Vitousek MN. 2017 Heritable variation in circulating glucocorticoids and endocrine flexibility in a free-living songbird. J. Evol. Biol. 30, 1724-1735. (doi:10.1111/jeb.13135)
61. Bairos-Novak KR, Ryan CP, Freeman AR, Anderson WG, Hare JF. 2018 Like mother, like daughter: heritability of female Richardson's ground squirrel Urocitellus richardsonii cortisol stress responses. Curr. Zool. 64, 153-163. (doi:10.1093/cz/zox014) ) and under selection, it would provide the basis for evolution to occur in a conserved higher-order mechanism that enables vertebrates to adjust their phenotype to fluctuations in climatic conditions [7,21,23,62] 7. Jessop TS, Lane ML, Teasdale L, Stuart-Fox D, Wilson RS, Careau V, Moore IT. 2016 Multiscale evaluation of thermal dependence in the glucocorticoid response of vertebrates. Am. Nat. 188, 342-356. (doi:10.1086/687588)
21. Malkoc K, Mentesana L, Casagrande S, Hau M. 2021 Quantifying glucocorticoid plasticity using reaction norm approaches: there still is so much to discover! Integr. Comp. Biol. 62, 58-70.
23. Ruuskanen S, Hsu B-Y, Nord A. 2021 Endocrinology of thermoregulation in birds in a changing climate. Mol. Cell. Endocrinol. 519, 111088. (doi:10.1016/j.mce.2020.111088)
62. Mentesana L, Hau M. 2022 Glucocorticoids in a warming world: do they help birds to cope with high environmental temperatures? Horm. Behav. 142, 105178. (doi:10.1016/j.yhbeh.2022.105178) .

Our prediction that corticosterone concentrations are higher at lower environmental temperatures at the population level was confirmed, in line with some earlier findings (summaries: [5–7] 5. Wingfield JC, Ramenofsky M. 2011 Hormone-behavior interrelationships of birds in response to weather. In Advances in the study of behavior, 43 (eds Brockmann HJ, Roper TJ, Naguib M, Mitani JC, Leigh WS), pp. 93-188. New York, NY: Academic Press.
6. de Bruijn R, Romero LM. 2018 The role of glucocorticoids in the vertebrate response to weather. Gen. Comp. Endocrinol. 269, 11-32. (doi:10.1016/j.ygcen.2018.07.007)
7. Jessop TS, Lane ML, Teasdale L, Stuart-Fox D, Wilson RS, Careau V, Moore IT. 2016 Multiscale evaluation of thermal dependence in the glucocorticoid response of vertebrates. Am. Nat. 188, 342-356. (doi:10.1086/687588) ). Importantly, we found that this negative relationship was explained by within-individual plasticity. A likely mechanism is increased energetic demands associated with thermoregulation that can lead to within-individual increases in corticosterone at lower temperatures (summaries: [23,62] 23. Ruuskanen S, Hsu B-Y, Nord A. 2021 Endocrinology of thermoregulation in birds in a changing climate. Mol. Cell. Endocrinol. 519, 111088. (doi:10.1016/j.mce.2020.111088)
62. Mentesana L, Hau M. 2022 Glucocorticoids in a warming world: do they help birds to cope with high environmental temperatures? Horm. Behav. 142, 105178. (doi:10.1016/j.yhbeh.2022.105178) ). In our study, temperatures at capture varied between approximately 4–27°C (electronic supplementary material, figures S1 and S2). Assuming that the lower limit of the thermoneutral zone in our great tit population is around 15°C [49,50], we collected two-thirds of the baseline and stress-induced corticosterone samples at temperatures that required the birds to produce heat to maintain body temperature. Two pathways underlying this response are conceivable. First, cooler environmental temperatures may have induced increases in corticosterone concentrations directly via thermoreceptors relaying information to hypothalamic brain areas (reviewed in [23,63] 23. Ruuskanen S, Hsu B-Y, Nord A. 2021 Endocrinology of thermoregulation in birds in a changing climate. Mol. Cell. Endocrinol. 519, 111088. (doi:10.1016/j.mce.2020.111088)
63. McKechnie AE. 2022 Regulation of body temperature: patterns and processes. In Sturkie's avian physiology (eds Scanes CG, Didri S), 7th edn, pp. 1211-1244. London, UK: Academic Press. ). Second, an indirect pathway could involve metabolic rate, which probably was higher in individuals that needed to generate heat through shivering and non-shivering thermogenesis [63]McKechnie AE. 2022 Regulation of body temperature: patterns and processes. In Sturkie's avian physiology (eds Scanes CG, Didri S), 7th edn, pp. 1211-1244. London, UK: Academic Press.. Indeed, experimentally induced increases in metabolic rate are positively associated with corticosterone concentrations within and among individual zebra finches (Taeniopygia guttata) [64,65] 64. Jimeno B, Hau M, Verhulst S. 2017 Strong association between corticosterone levels and temperature-dependent metabolic rate in individual zebra finches. J. Exp. Biol. 220, 4426-4431. (doi:10.1242/jeb.166124)
65. Jimeno B, Hau M, Verhulst S. 2018 Corticosterone levels reflect variation in metabolic rate, independent of ‘stress’. Sci. Rep. 8, 13020. (doi:10.1038/s41598-018-31258-z) . Metabolic rates may also have been increased at lower temperatures because great tits had to exhibit greater foraging activity to provision their young as insect food is harder to find and less abundant (e.g. [66]66. Avery MI, Krebs JR. 1984 Temperature and foraging success of great tits Parus major hunting for spiders. Ibis. 126, 33-38. (doi:10.1111/j.1474-919X.1984.tb03661.x) ).

At present, we cannot exclude that other factors associated with environmental temperature, for example, precipitation, wind, or barometric pressure contributed to within-individual corticosterone plasticity [5,6] 5. Wingfield JC, Ramenofsky M. 2011 Hormone-behavior interrelationships of birds in response to weather. In Advances in the study of behavior, 43 (eds Brockmann HJ, Roper TJ, Naguib M, Mitani JC, Leigh WS), pp. 93-188. New York, NY: Academic Press.
6. de Bruijn R, Romero LM. 2018 The role of glucocorticoids in the vertebrate response to weather. Gen. Comp. Endocrinol. 269, 11-32. (doi:10.1016/j.ygcen.2018.07.007) . Furthermore, because most of the repeated sampling occurred across years, variables unrelated to environmental temperature could also have contributed to within-individual plasticity in corticosterone. These include an individual's experiences within a year that may have altered its perception, processing and responses to environmental challenges, for example via epigenetic modifications of both HPA axis (and thus corticosterone release) and receptor functioning (e.g. [67,68] 67. Turecki G, Meaney MJ. 2016 Effects of the social environment and stress on glucocorticoid receptor gene methylation: a systematic review. Biol. Psychiatry 79, 87-96. (doi:10.1016/j.biopsych.2014.11.022)
68. Jimeno B, Hau M, Gómez-Díaz E, Verhulst S. 2019 Developmental conditions modulate DNA methylation at the glucocorticoid receptor gene with cascading effects on expression and corticosterone levels in zebra finches. Sci. Rep. 9, 15869. (doi:10.1038/s41598-019-52203-8) ). To address these questions, future work should sample individuals at shorter time scales like across seasons, life-history stages, or even days that vary in environmental temperatures. Moreover, experimental manipulations of environmental temperatures and/or an individual's thermoregulatory abilities will be important to ascertain causal relationships.

As expected, individuals in our study also differed from each other in average baseline and stress-induced concentrations (i.e. reaction norm elevation, table 2 and figure 1). Our corticosterone repeatability estimates (baseline: 17%; stress-induced: 26%) are within the range of those reported in recent meta-analyses (across vertebrate taxa, baseline: 18–23%; stress-induced: 37–38%; [47,48] 47. Schoenemann KL, Bonier F. 2018 Repeatability of glucocorticoid hormones in vertebrates: a meta-analysis. PeerJ. 6, e4398. (doi:10.7717/peerj.4398)
48. Taff CC, Schoenle LA, Vitousek MN. 2018 The repeatability of glucocorticoids: a review and meta-analysis. Gen. Comp. Endocrinol. 260, 136-145. (doi:10.1016/j.ygcen.2018.01.011) ). At the same time, our estimates suggest that a substantial percentage of the variance remains unexplained (residual variance), which could in principle result from high within-individual variation, low among-individual variation, or high measurement error acting singly or in combination [47]47. Schoenemann KL, Bonier F. 2018 Repeatability of glucocorticoid hormones in vertebrates: a meta-analysis. PeerJ. 6, e4398. (doi:10.7717/peerj.4398) . Given our long sampling intervals, we consider changes in environmental or internal conditions of an individual a likely source of high residual variance, as shorter sampling intervals tend to increase repeatability estimates for glucocorticoid traits [47,48] 47. Schoenemann KL, Bonier F. 2018 Repeatability of glucocorticoid hormones in vertebrates: a meta-analysis. PeerJ. 6, e4398. (doi:10.7717/peerj.4398)
48. Taff CC, Schoenle LA, Vitousek MN. 2018 The repeatability of glucocorticoids: a review and meta-analysis. Gen. Comp. Endocrinol. 260, 136-145. (doi:10.1016/j.ygcen.2018.01.011) . Nevertheless, our repeatability estimates align well with heritability estimates in studies on free-living bird populations, where additive genetic effects explained less than 20% of the variance in baseline and more than 30% in stress-induced concentrations, respectively [60,69,70] 60. Stedman JM, Hallinger KK, Winkler DW, Vitousek MN. 2017 Heritable variation in circulating glucocorticoids and endocrine flexibility in a free-living songbird. J. Evol. Biol. 30, 1724-1735. (doi:10.1111/jeb.13135)
69. Jenkins BR, Vitousek MN, Hubbard JK, Safran RJ. 2014 An experimental analysis of the heritability of variation in glucocorticoid concentrations in a wild avian population. Proc. R. Soc. B 281, 20141302. (doi:10.1098/rspb.2014.1302)
70. Béziers P, San-Jose LM, Almasi B, Jenni L, Roulin A. 2019 Baseline and stress-induced corticosterone levels are heritable and genetically correlated in a barn owl population. Heredity 123, 337-348. (doi:10.1038/s41437-019-0203-5) .

The presence of I × E in our great tit population, i.e. among-individual differences corticosterone plasticity at both levels (figure 1, table 3), is intriguing from a proximate viewpoint. In principle, individual differences in plasticity (reaction norm slope) could arise from two non-exclusive processes: (i) long-term individual differences that are based on genetic information, maternal or other early developmental effects; or (ii) short-term variation in an individual's state—or both (e.g. [71,72] 71. Sih A, Mathot KJ, Moirón M, Montiglio P-O, Wolf M, Dingemanse NJ. 2015 Animal personality and state–behaviour feedbacks: a review and guide for empiricists. Trends Ecol. Evol. 30, 50-60. (doi:10.1016/j.tree.2014.11.004)
72. Dingemanse NJ, Wolf M. 2013 Between-individual differences in behavioural plasticity within populations: causes and consequences. Anim. Behav. 85, 32. (doi:10.1016/j.anbehav.2012.12.032) ). Although we sampled great tits during a standardized parental phase, individuals could differ in various aspects, including body condition, health status, territory or partner quality, predation pressure, reproductive investment and age (sexes differed in some aspects, see table 3; electronic supplementary material, table S3, but sample size limitations did not allow us to test whether sexes differed in plasticity). Both long- and short-term processes could generate among-individual differences in glucocorticoid slopes via variation in, for example, the perception and neuro-endocrine processing of environmental temperature, the capacity to synthesize glucocorticoids (including the sensitivity of the HPA axis to releasing hormones), the binding of glucocorticoids to carrier molecules in the blood, their enzymatic deactivation, and differences in receptor densities [13,14,73] 13. Hau M, Casagrande S, Ouyang JQ, Baugh AT. 2016 Glucocorticoid-mediated phenotypes in vertebrates: multilevel variation and evolution. In Advances in the study of behavior, 48 (eds Naguib M, Mitani JC, Simmons LW, Barrett L, Healy S, Zuk M), pp. 41-115. New York, NY: Academic Press.
14. Taff CC, Vitousek MN. 2016 Endocrine flexibility: optimizing phenotypes in a dynamic world? Trends Ecol. Evol. 31, 476-488. (doi:10.1016/j.tree.2016.03.005)
73. Wingfield JC. 2013 Ecological processes and the ecology of stress: the impacts of abiotic environmental factors. Funct. Ecol. 27, 37-44. (doi:10.1111/1365-2435.12039) . Many short- or long-term variables can also determine an individual's energy intake and/or usage and thus affecting circulating glucocorticoid concentrations [35,74] 35. Romero LM, Dickens MJ, Cyr NE. 2009 The reactive scope model – a new model integrating homeostasis, allostasis, and stress. Horm. Behav. 55, 375-389. (doi:10.1016/j.yhbeh.2008.12.009)
74. McEwen BS, Wingfield JC. 2003 The concept of allostasis in biology and biomedicine. Horm. Behav. 43, 2-15. (doi:10.1016/S0018-506X(02)00024-7) . These hypotheses can be tested experimentally.

Elevation and slope of reaction norms in baseline corticosterone were correlated within individuals, suggesting that these two features of the endocrine response to environmental temperature are linked in our free-living great tit population (table 2). The fanning-in pattern evident in baseline corticosterone slopes (figure 1a) suggests a reduction (canalization) in phenotypic variance at higher temperatures. At present, we can only speculate that differences in the individuals' energetic or health state may play a role. Glucocorticoids have context-, state- and tissue-dependent effects: they can mobilize energy reserves in adipose tissue but also increase fat storage in the liver (reviewed in [31]Landys MM, Ramenofsky M, Wingfield JC. 2006 Actions of glucocorticoids at a seasonal baseline as compared to stress-related levels in the regulation of periodic life processes. Gen. Comp. Endocrinol. 148, 132-149. (doi:10.1016/j.ygcen.2006.02.013) ). Thus, future work needs to assess an individual's energetic state (e.g. via morphometric, or blood and tissue composition measures), ideally coupled with a manipulation of energetic challenges. From an evolutionary viewpoint, the covariation between elevation and slope is important because it suggests that these two attributes of baseline corticosterone are somehow linked [21]21. Malkoc K, Mentesana L, Casagrande S, Hau M. 2021 Quantifying glucocorticoid plasticity using reaction norm approaches: there still is so much to discover! Integr. Comp. Biol. 62, 58-70. .

Finally, great tit individuals showed a negative relationship in both baseline and stress-induced corticosterone with environmental temperature (table 3), suggesting a concerted response in these two traits to the same environmental factors. The positive covariation between baseline and stress-induced corticosterone at the population, among-individual and within-individual level (electronic supplementary material, table S5) supports this view. A likely scenario is that environmental temperature affects an individual's allostatic load or reactive homeostasis (i.e. the cumulative challenges, energetic or otherwise, that an individual has to cope with at one point in time; [35,74] 35. Romero LM, Dickens MJ, Cyr NE. 2009 The reactive scope model – a new model integrating homeostasis, allostasis, and stress. Horm. Behav. 55, 375-389. (doi:10.1016/j.yhbeh.2008.12.009)
74. McEwen BS, Wingfield JC. 2003 The concept of allostasis in biology and biomedicine. Horm. Behav. 43, 2-15. (doi:10.1016/S0018-506X(02)00024-7) ), and this change in state could influence both traits—also in different populations and species (e.g. [34,75] 34. Vitousek MN et al.. 2019 Macroevolutionary patterning in glucocorticoids suggests different selective pressures shape baseline and stress-induced levels. Am. Nat. 193, 866-880.
75. Zimmer C, Taff CC, Ardia DR, Rose AP, Aborn DA, Johnson LS, Vitousek MN. 2020 Environmental unpredictability shapes glucocorticoid regulation across populations of tree swallows. Sci. Rep. 10, 13682. (doi:10.1038/s41598-020-70161-4) Crossref, PubMed, ISI, Google Scholar ).

05 - Conclusion

This line of work is important for evaluating the consequences of climate change for wild vertebrate populations. Our reaction norm study suggests that individuals from a wild bird population plastically change glucocorticoid concentrations in response to a key environmental gradient – temperature – and that they differ in the extent of their glucocorticoid plasticity. These results are vital for a refined understanding of the physiological processes that underlie animal–environment interactions as well as the evolutionary forces that may shape them. As next steps, we should test the generality of these findings for a wider range of species and environmental factors and connect individual variation in glucocorticoid reaction norm components to expressed phenotypes like reproductive success, recruitment and survival, and study patterns of selection [13–15,21] 13. Hau M, Casagrande S, Ouyang JQ, Baugh AT. 2016 Glucocorticoid-mediated phenotypes in vertebrates: multilevel variation and evolution. In Advances in the study of behavior, 48 (eds Naguib M, Mitani JC, Simmons LW, Barrett L, Healy S, Zuk M), pp. 41-115. New York, NY: Academic Press. Google Scholar
14. Taff CC, Vitousek MN. 2016 Endocrine flexibility: optimizing phenotypes in a dynamic world? Trends Ecol. Evol. 31, 476-488. (doi:10.1016/j.tree.2016.03.005) Crossref, PubMed, ISI, Google Scholar
15. Wada H, Sewall KB. 2014 Introduction to the symposium—uniting evolutionary and physiological approaches to understanding phenotypic plasticity. Integr. Comp. Biol. 54, 774-782. (doi:10.1093/icb/icu097) Crossref, PubMed, ISI, Google Scholar
21. Malkoc K, Mentesana L, Casagrande S, Hau M. 2021 Quantifying glucocorticoid plasticity using reaction norm approaches: there still is so much to discover! Integr. Comp. Biol. 62, 58-70. Crossref, ISI, Google Scholar . Moreover, the integration of plastic glucocorticoid responses with multiple other traits will be a rewarding future avenue, as it has the potential to provide important insights into the evolutionary patterns underlying multivariate plasticity (i.e. ‘integration of plasticity’, [21,76] 21. Malkoc K, Mentesana L, Casagrande S, Hau M. 2021 Quantifying glucocorticoid plasticity using reaction norm approaches: there still is so much to discover! Integr. Comp. Biol. 62, 58-70. Crossref, ISI, Google Scholar
76. Pigliucci M. 2001 Characters and environments. In The character concept in evolutionary biology (ed. Wagner GP), pp. 363-388. New York, NY: Academic Press. Crossref, Google Scholar ), and overall, the evolution of reversible phenotypes. Although reaction norm studies require large sample sizes both in number of individuals measured and observations per individual, other areas in biology can also draw insights from this work. For example, the integration of reaction norms into animal welfare, evolutionary medicine and public health concepts may lead to more nuanced policies and improved treatments [77–79] 77. Wells JCK, Nesse RM, Sear R, Johnstone RA, Stearns SC. 2017 Evolutionary public health: introducing the concept. Lancet 390, 500-509. (doi:10.1016/S0140-6736(17)30572-X) Crossref, PubMed, ISI, Google Scholar
78. Gurven MD, Lieberman DE. 2020 WEIRD bodies: mismatch, medicine and missing diversity. Evol. Hum. Behav. 41, 330-340. (doi:10.1016/j.evolhumbehav.2020.04.001) Crossref, ISI, Google Scholar
79. Arndt SS, Goerlich VC, van der Staay FJ. 2022 A dynamic concept of animal welfare: the role of appetitive and adverse internal and external factors and the animal's ability to adapt to them. Front. Anim. Sci. 3. (doi:10.3389/fanim.2022.908513) Crossref, Google Scholar .

Ethics

All field procedures were conducted in compliance with the legal requirements of Germany and were approved by the cognizant regional governmental authority, the Regierungspräsidium von Oberbayern, Germany (license no. ROB-55.2-2532-Vet_02-13-204 & ROB-55.2-2532-Vet_02-15-25).

Data Accessibility

The dataset and the R code generated for this study are available on Zenodo: https://doi.org/10.5281/zenodo.7018713 [80]80. Hau M, Deimel C, Moiron M. 2022 Data and code from: Great tits differ in glucocorticoid plasticity in response to spring temperature (v1.0.1). Data set. Zenodo. (doi:10.5281/zenodo.7018713) Google Scholar. An earlier version of this manuscript was posted at bioRxiv: https://doi.org/10.1101/2022.04.21.489013 [81]81. Moiron M. 2022 Great tits differ in glucocorticoid plasticity in response to spring temperature. bioRxiv. (doi:10.1101/2022.04.21.489013) Google Scholar. Additional information is available in the electronic supplementary material [82]82. Hau M, Deimel C, Moiron M. 2022 Great tits differ in glucocorticoid plasticity in response to spring temperature. Figshare. (doi:10.6084/m9.figshare.c.6261884) Google Scholar.

Conflict of interest

The authors declare that no competing interests exist.

Funding

This work was funded by the Max Planck Society. M.M. was funded by an Alexander von Humboldt research fellowship.

Acknowledgments

We are indebted to Sabine Jörg and Robert de Bruijn for their field and laboratory help, and to Julia Cramer, Natalia Perez-Ruiz, Nataly Hidalgo Aranzamendi, Sam Hardman, Holland Galante and Skylar Buckingham for field assistance. Many thanks to the Erzdiozöse München und Freising, especially K. Meindl, B. Vollmar and M. Laußer for allowing us to work in their forest. The Hau-Goymann laboratory group provided input that greatly improved data collection and manuscript preparation.

Authors' Contribution

M.H.: conceptualization, data curation, funding acquisition, investigation, methodology, project administration, resources, supervision, validation, writing—original draft, writing—review and editing; C.D.: data curation, investigation, validation, writing—review and editing; M.M.: data curation, formal analysis, investigation, methodology, software, visualization, writing—review and editing.

All authors gave final approval for publication and agreed to be held accountable for the work performed therein.

Footnotes

Electronic supplementary material is available online at https://doi.org/10.6084/m9.figshare.c.6261884.

© 2022 The Authors.

Published by the Royal Society under the terms of the Creative Commons Attribution License https://creativecommons.org/licenses/by/4.0/, which permits unrestricted use, provided the original author and source are credited.