Telomere dynamics in parasitic great spotted cuckoos and their magpie hosts

Telome e dynamics in pa asi ic g ea spo ed cuckoos and hei magpie hos s J. J. SOLER*, C. RUIZ CASTELLANO*, J. MARTI ´NEZ-DE LA PUENTE†,G.TOM  AS*, M. RUIZ-RODRI ´GUEZ* & J. FIGUEROLA† *Depa amen o de Ecolog ıa Funcional y E olu i a, Es aci on Expe imen al de Zonas  A idas (CSIC), Alme  ıa, Spain †Depa amen o de Ecolog ıa de Humedales, Es aci on Biol ogica Do~ nana (CSIC), Se illa, Spain Keywo ds: b ood pa asi ism; cell di ision; incuba ion pe iod; li e his o y; nes ling g ow h; oxida i e damage; elome ase ac i i y. Abs ac Al hough li le is known on he impac o en i onmen on elome e leng h dynamics, i has been sugges ed o be a ec ed by s ess, li es yle and/o li e- his o y s a egies o animals. We he e compa ed elome e dynamics in e y- h ocy es o ha chlings and ledglings o he b ood pa asi e g ea spo ed cuckoos (Clama o glanda ius) and o magpies (Pica pica), hei main hos in Eu ope. In magpie chicks, elome e leng h dec eased om ha ching o ledg- ing, whe eas no signi ican change in elome e leng h o g ea spo ed cuckoo chicks was ound. Mo eo e , we ound in e speci ic di e ences in he associa ion be ween laying da e and elome e sho ening. In e speci ic di e ences in elome e sho ening we e in e p e ed as a consequence o di - e ences in li es yle and li e-his o y cha ac e is ics o magpies and g ea spo - ed cuckoos. In compa ison wi h magpies, cuckoos expe ience educed sibling compe i ion and highe access o esou ces and, consequen ly, lowe s ess ul en i onmen al condi ions du ing he nes ling phase. These cha ac- e is ics also explain he associa ions be ween elome e a i ion and en i- onmen al condi ions (i.e. laying da e) o magpies and he absence o associa ion o g ea spo ed cuckoos. These esul s he e o e i expec a ions on elome e dynamics de i ed om in e speci ic di e ences in li es yle and li e his o y o b ood pa asi es and hei bi d hos s. In oduc ion En i onmen al condi ions expe ienced du ing he i s days o li e may ha e impo an consequences la e , du ing adul hood, in e ms o su i al p ospec s (Mon- aghan, 2008). Recen ly, i has been sugges ed ha hese delayed e ec s a e a leas pa ially d i en by he in lu- ence o ea ly en i onmen al condi ions on elome e dynamics (Bee y e al., 2012) and suppo ing e idences ha e been accumula ed du ing he las ew yea s (Hall e al., 2004; Heidinge e al., 2012; Boonekamp e al., 2014; He bo n e al., 2014; Ne le e al., 2015). Telom- e es a e specialized s uc u es a he end o he ch omo- some, which consis on sho s epea s o he noncoding DNA sequence TTAGGG ha , among o he unc ions, p o ec wholeness o gene ic in o ma ion du ing cell di ision (Blackbu n, 1991). Telome e a i ion has adi- ionally been ela ed wi h ageing (Sahin & DePinho, 2010) and is closely ela ed o s ess e en s o di e en na u e ha include hose associa ed wi h cell di ision (i.e. g ow h) and wi h ele a ed concen a ions o eac- i e oxygen species (Monaghan & Haussmann, 2006). Ageing, li es yle, li espan and li e-his o y cha ac e is- ics o animals a e closely ela ed in na u e (Monaghan & Haussmann, 2006); o example, animals wi h ela- i e sho li espan g ow as e and ep oduce ea lie a he cos o inc easing me abolic and cellula degene a- ion a es (Rickle s & Finch, 1995), which would a ec elome e dynamics. Telome e leng h and dynamics a e conside ed good indica o s o i ness in e ms o su - i al p ospec o he adop ed li es yle and/o li e-his o y s a egies (Monaghan & Haussmann, 2006; Ko schal e al., 2007). Thus, de ec ing e idence linking li es yle and/o li espan wi h elome e leng h and sho ening is o p ime impo ance o unde s and he balance o cos s Co espondence: Juan J. Sole , Depa amen o de Ecolog ıa Funcional y E olu i a, Es aci on Expe imen al de Zonas  A idas (CSIC), C a. Sac amen o s/n, La Ca~ nada de San U bano, Alme  ıa, Spain. Tel.: +34 950281045; ax: +34 950277100; e-mail: [email p o ec ed] ª2015 EUROPEAN SOCIETY FOR EVOLUTIONARY BIOLOGY. J. EVOL. BIOL. 1 JOURNAL OF EVOLUTIONARY BIOLOGY ª2015 EUROPEAN SOCIETY FOR EVOLUTIONARY BIOLOGY doi: 10.1111/jeb.12680 and bene i s associa ed wi h species-speci ic li e his o- ies (Salomons e al., 2009b; Monaghan, 2014). Pa icu- la ly in e es ing is he s udy o elome e dynamics du ing he i s ew days o li e o al icial bi ds o se - e al easons. Na u al selec ion and elome e sho ening is pa icula ly appa en a his s age due o e y high cell di ision a e (Salomons e al., 2009b). Mo eo e , he ela i ely down- egula ion o elome ase ac i i y (i.e. educed elome e epai ) du ing he exponen ial g ow h s age (Fo sy h e al., 2002; bu see Haussmann e al., 2007) makes easie he de ec ion o changes in elome e leng h o explo e he p edic ed associa ions wi h li e-his o y cha ac e is ics o en i onmen al condi- ions. In addi ion, en i onmen al condi ions expe ienced by nes lings la gely a y in associa ion wi h laying da e, wi h la e ha chlings ypically de eloping in wo se, mo e s ess ul condi ions han ea ly ha chlings, due o poo pa en al quali y o b eede s and/o de e io a ed esou ce a ailabili y (De Ne e e al., 2004; Ve huls & Nilsson, 2008). Fu he , he s eng h o he associa ion be ween laying da e and esou ce a ailabili y o de el- oping nes lings depends on species-speci ic li e-his o y ai s (S ensson, 1995) and, hus, we should expec elome e dynamics o a y wi h laying da e (Foo e e al., 2011), a leas o some species. As a as we know, his p edic ed associa ion has ne e been es ed in he wild. Finally, sibling compe i ion o pa en al ca e is one impo an cause o physiological s ess (Ma  ınez-Padilla e al., 2004; Blanco e al., 2006) likely a ec ing elome e dynamics du ing g ow h. Consequen ly, p e ious s ud- ies ha e ound ha he s eng h o in ab ood compe i- ion o ood in jackdaw (Co us monedula) (Salomons e al., 2009a) and Eu opean s a ling (S u nus ulga is) (Ne le e al., 2013) nes lings was posi i ely ela ed wi h elome e a i ion du ing hei i s ew days o li e. Thus, because he s eng h o sibling compe i ion widely a ies wi hin and among bi d species (Mock & Pa ke , 1997), i can be expec ed ha nes lings o spe- cies wi h in ense sibling i al y had be e mechanisms o cope wi h s ess and o educe elome e sho ening, han hose o species wi h low sibling compe i ion. A he in aspeci ic le el, elome e leng h and body size o ba n swallow (Hi undo us ica) nes lings posi i ely co a ied (Cap ioli e al., 2013), and elome e a i ion due o sibling compe i ion was nega i ely ela ed wi h nes ling hie a chy in Eu opean s a lings (Ne le e al., 2013, 2015); bo h esul s sugges ing he possibili y o physiological adjus men depending on nes ling hie a - chy (i.e. body mass) ha could e en be media ed by ma e nal e ec s. Mo eo e , expe imen ally inc eased b ood size also esul ed in la ge elome e a i ion in zeb a inch (Taeniopygia gu a a) pa en s (Reiche e al., 2014). Thus, species di e ing in he s eng h o in a- b ood compe i ion o ood may also di e in elome e dynamics du ing g ow h. Wi h he aim o con ibu ing o unde s and ac o s explaining elome e dynamics o de eloping animals, we he e explo ed he associa ion be ween elome e leng h o ha chlings and ledglings o magpies (Pica pica), and o hei b ood pa asi ic g ea spo ed cuckoos (Clama o glanda ius), in ela ion o laying da e. Explo - ing hese associa ions in his b ood pa asi e–hos sys em has se e al ad an ages. Fi s , magpies a e he main Eu opean hos o g ea spo ed cuckoos, and he e o e, nes lings o bo h species a e ea ed by magpie adul s (Sole e al., 1999; Sole & Sole , 2000). Second, en i- onmen al condi ions expe ienced by de eloping g ea spo ed cuckoo and magpie nes lings usually di e , he la e de eloping in a mo e compe i i e en i onmen han he o me . G ea spo ed cuckoo nes lings usually ou compe e os e siblings soon a e ha ching. Mo e- o e , pos -ha ching g ow h a e o g ea spo ed cuck- oos is much highe han ha o magpie hos s (Sole & Sole , 1991). Consequen ly, i elome e dynamics depend on cell di ision a es, ha o g ea spo ed cuck- oos and magpies should di e . Finally, because g ea spo ed cuckoo nes lings usually sha e he nes s wi h ewe chicks han magpies do, i is likely ha he expec ed e ec s o en i onmen al condi ions ela ed o esou ce a ailabili y (i.e. laying da e (De Ne e e al., 2004)) on elome e dynamics a e weake o nes lings o he b ood pa asi ic species. Ma e ials and me hods S udy si es and ieldwo k The s udy was pe o med in he Guadix (37°180N, 3°110W, sou he n Spain) magpie popula ion, si ua ed in a high-al i ude pla eau (app ox. 1000 m a.s.l.). The ege a ion o he a ea is spa se, including uncul i a ed a eas and many g o es o almond (P unus dulcis) and pine (Pinus halepensis and Pinus pinas e ) ees and oaks (Que cus o undi olia) in which magpies p e e o build hei nes s. B ood pa asi ism by g ea spo ed cuckoos is qui e common in he a ea (Sole e al., 2001, 2013) and, al hough g ea spo ed cuckoos usually ou com- pe e magpie os e siblings (Sole & De Ne e, 2013), pa asi ic nes lings do no e ic hos nes lings a e ha ching and, he e o e, bo h species may g ow up oge he in he same nes (Ma  ın-G al ez e al., 2011). Fieldwo k was conduc ed du ing he 2013 b eeding season. A he beginning o sp ing in Ma ch–Ap il, we in ensely sea ched o nes s o magpies ha we e e is- i ed egula ly o de e mine hei laying da e. Expec ed ha ching da e o magpies and g ea spo ed cuckoos was es ima ed as 25 and 19 days, espec i ely, a e laying da e o he i s magpie egg. Magpie nes s we e hen isi ed du ing hese and subsequen days up o sam- pling magpie ha chlings (i.e. 0–1 day old) and, in he case o pa asi ized nes s, g ea spo ed cuckoo ha ch- lings. Because ma e nal and gene ic ac o s a e known ª2015 EUROPEAN SOCIETY FOR EVOLUTIONARY BIOLOGY. J. EVOL. BIOL. doi: 10.1111/jeb.12680 JOURNAL OF EVOLUTIONARY BIOLOGY ª2015 EUROPEAN SOCIETY FOR EVOLUTIONARY BIOLOGY 2JUAN J. SOLER ET AL. o de e mine ea ly li e elome e leng h (Asgha e al., 2015b), we collec ed a d op o blood om he b achial ein o wo andomly selec ed magpie ha chlings and om all cuckoo ha chlings in a magpie nes . All mag- pies and g ea spo ed cuckoo nes lings in he nes s 18 and 16 days a e ha ching, espec i ely, we e again blood-sampled. To inc ease he numbe o pa asi ized nes s wi h magpies and g ea spo ed cuckoo ledglings g owing up oge he , we expe imen ally pa asi ized 11 magpie nes s wi h a single cuckoo egg close o ha ching ime om mul ipa asi ized magpie nes s. In h ee o hese nes s, only he cuckoo ledged [cuckoos ha ched 3.33 (SE =0.67) days be o e magpies]; in one, only magpies ( ou magpie ledglings ha ha ched 2 days in ad ance o cuckoos); and in se en nes s, bo h he g ea spo ed cuckoo and magpies (a e age numbe o ledglings pe nes =2.6, SD =0.98) ledged [cuckoos ha ched 2.14 (SE =0.86) days be o e magpies]. In addi ion, we sam- pled 27 na u al magpie nes s. F om eigh o hese nes s, only cuckoos ledged (mean =1.13, SD =0.35), om 18 only magpies ledged (mean =4.5, SD =1.04), and om he las nes , one g ea spo ed cuckoo and one magpie success ully eached he ledging s age. How- e e , duplica ed es ima es o elome e leng h o some samples we e no consis en ly simila (i.e. CV >5%, see below) and we e no conside ed in he analyses. We hus collec ed in o ma ion wi h adequa e es ima es o elome e a i ion o 36 magpie nes s. In eigh o hese nes s, magpies and g ea spo ed cuckoos g ew oge he , bu in o ma ion o magpies and cuckoo nes - lings was only a ailable o ou o hem. Fo he o he ou nes s wi h bo h species, only in o ma ion o mag- pie nes lings was a ailable o he analyses. In addi ion, we collec ed in o ma ion on elome e a i ion o 18 and 10 nes s, espec i ely, whe e only chicks o one species, ha is ei he magpies o g ea spo ed cuckoos, g ew up. Fo some addi ional nes s, in o ma ion o elome e leng h was only a ailable o ha chling o o ledglings. Thus, o cuckoo nes lings, inal sample size was 18 o elome e leng h o ha chling and 14 o elome e a i ion. Fo magpie nes lings, inal sample size used in he analyses was 26 and 27 o elome e leng h o ha chlings and ledglings, espec i ely, and 26 o elome e a i ion. Labo a o y wo k DNA was ex ac ed om blood samples using a s an- da d chlo o o m–isoamyl alcohol-based p o ocol (see Fe agu i e al., 2013). DNA concen a ion was adjus ed o 20 ng lL 1 using dis illed wa e and conse ed o- zen un il u he analyses. Rela i e elome e leng h (he ea e elome e leng h) was es ima ed by q-PCR ol- lowing he p o ocol and p ime s o C iscuolo e al. (2009). As con ol single-copy gene we used he glyce - aldehyde-3-phospha e dehyd ogenase (GAPDH). GAPDH was used o no malize he quan i y o elome e sequence o he amoun o DNA in he q-PCR. The inal PCR olume was 20 lL con aining 10 lLo Ligh Cycle 480 SYBR G een I Mas e (Roche Diagnos- ics GmbH, Mannheim, Ge many) and 1 lL o DNA a 20 ng lL 1 o DNA. The eac ions o elome es o GAPDH we e done in di e en pla es due o he di e - en ial PCR condi ions. Telome e PCR condi ions we e 10 min a 95 °C ollowed by 30 cycles o 1 min a 56 °C and 1 min a 95 °C. GAPDH PCR s a ed wi h 10 min a 95 °C ollowed by 40 cycles o 1 min a 60 °C and 1 min a 95 °C, bo h pe o med in a Ligh Cycle 480 RT-PCR Sys em (Roche). Each sample was un in duplica e, and samples wi h a coe icien o a ia ion highe o 5% we e emo ed om he analy- ses. Each 96-well pla e included se ial dilu ions o DNA (40 ng, 10 ng, 2.5 ng, 0.66 ng o DNA pe well) om a e e ence pool ( he in e nal con ol) un in iplica e, which we e used o gene a e he s anda d cu es, and a blank con ol wi h no DNA. Quan i ica ion cycle al- ues (C ) we e ans o med in o no malized ela i e quan i ies (NRQs) ollowing Hellemans e al. (2007) p ocedu e, which con ols o he ampli ying e iciency o each qPCR. Ampli ica ion e iciency o elome e p oduc s anged be ween 1.85 and 2.01 and o he GAPDH p oduc be ween 1.88 and 2.14. The slope o he calib a ion cu e anged be ween -3.792 and -3.277 o he elome e p oduc and -3.647 and -3.031 o he GAPDH p oduc . The mel ing cu es o he con ol gene cycles con i med no e idence o p ime dime o non- speci ic ampli ica ion. Di e en echniques a e a ailable o measu ing elome e leng h in wildli e ( e iewed in Nussey e al., 2014). Each o hese echniques has ad an ages and limi a ions ha may a ec he eliabil- i y o he esul s. In he case o q-PCR, an impo an limi a ion is ha in e s i ial elome ic sequences may occu on he genome and consequen ly may bias es i- ma es o ela i e elome e leng h. Consequen ly, q-PCR es ima ed elome e leng hs may be no adequa e o compa e elome e leng h among species (Nussey e al., 2014). Howe e , i is adequa e o compa e pa e ns o a ia ion ac oss species based on epea ed measu es o he same indi iduals ac oss ime, as we ha e done in his s udy ( o a simila app oach, see Asgha e al., 2015a). Consequen ly, we will no deal wi h in e speci- ic di e ences in elome e leng h bu compa e he em- po al and de elopmen al pa e ns o a ia ion in elome e leng h be ween magpies and cuckoos. S a is ical analyses F equency dis ibu ion o NRQ alues es ima ed o nes lings and ledglings o cuckoos and magpies did no di e om no mal dis ibu ions (Kolmogo o –Smi no es o con inuous a iables, P>0.05). Mo eo e , be ween-nes a ia ion in elome e leng h o magpie ledglings was signi ican ly la ge han wi hin-nes a i- ª2015 EUROPEAN SOCIETY FOR EVOLUTIONARY BIOLOGY. J. EVOL. BIOL. doi: 10.1111/jeb.12680 JOURNAL OF EVOLUTIONARY BIOLOGY ª2015 EUROPEAN SOCIETY FOR EVOLUTIONARY BIOLOGY B ood pa asi ism and elome e dynamics 3 a ion (R=0.67, F 26,77 =4.99, P<0.0001); hus, we used wi hin-nes mean alues o subsequen analyses. Telome e a i ion was hus es ima ed as he di e ence be ween a e age alues a he ime o ha ching and a he ime o ledging. Laying da e did no di e om no mal dis ibu ion (Kolmogo o –Smi no es o con- inuous a iables, P>0.05), and hus, we used pa a- me ic s a is ical es s o compa ing elome e leng h and a i ion o g ea spo ed cuckoos and magpies and i s ela ionship wi h hei espec i e laying da e. Whe he o no cuckoo o magpie chicks g ew up oge he wi h nes lings o he o he species did no a ec elome e leng h and elome e a i ion o ledgling cuck- oos [sample size: N(only cuckoos) =10, N(cuckoos and magpies) =4, <1.02, P>0.33] o o magpie nes lings [sample size: N(only magpies) =19, N(cuckoos and magpies) =8, <0.35, P>0.73]. Thus, his ac o was no conside ed in u he analyses. Some nes lings o bo h species died la e du ing he nes ling phase be o e he second blood sampling, and hus, sample sizes o g ea spo ed cuckoos and magpies in pa asi ized nes s ea ing nes lings o bo h species do no coincide. Telome e dynamics o g ea spo ed cuckoos and mag- pies we e i s explo ed sepa a ely o each species by means o epea ed-measu es ANOVAs wi h es ima es om he same nes a di e en imes as wi hin ac o and b ood size and laying da e as co a iables. In e speci ic di e - ences in he associa ion be ween laying da e and ei he elome e leng h a di e en nes s age (i.e. ha ching and ledging) o elome e sho ening we e explo ed wi h AN- COVAs wi h species iden i y as he ca ego ical independen ac o , laying da e o he i s magpie egg as he co a iable and he in e ac ion be ween laying da e and species iden- i y as he ac o in o ming on he in e speci ic di e - ences. Rela ionships be ween laying da e and ei he elome e leng h o elome e sho ening we e explo ed using Pea son co ela ions. All s a is ical es s we e pe o med in S a is ica 10.0 (S a so Inc., 2011). Resul s As magpie nes lings expe ienced a ela i ely d as ic elome e sho ening du ing he nes ing pe iod ( epea ed-measu es ANOVA,F 1,25 =45.65, P<0.0001, Fig. 1), his was no he case o cuckoo nes lings ( epea ed-measu es ANOVA,F 1,13 =0.19, P=0.67, Fig. 1; in e speci ic compa isons o elome e sho ening, in e - ac ion be ween de eloping ime and species iden i y, F 1,38 =16.68, P=0.0002, Fig 1). O e all, elome e leng h o magpies declined by 64.3% om ha ching o ledging. Including he nonsigni ican e ec s o laying da e and b ood size (P>0.28) in he s a is ical models explo ing in e speci ic di e ences did no change any conclusions. The associa ion be ween elome e leng h and laying da e di e ed o g ea spo ed cuckoo and magpie ha chlings (ANCOVA, in e ac ion be ween species iden i y and laying da e, F 1,40 =7.48, P=0.009, Fig. 2). Telom- e e leng h o magpie ha chlings showed a dec ease in ela ion o laying da e (R=0.44, =2.37, N=26, P=0.026, Fig. 2), whe eas elome e leng h o cuckoo ha chlings was posi i ely ela ed o laying da e (R=0.49, =2.28, N=18, P=0.037, Fig. 2). A he ime o ledging, he associa ion be ween elom- e e leng h and laying da e o g ea spo ed cuckoos and magpies also di e ed signi ican ly (ANCOVA, in e ac ion be ween species iden i y and laying da e, F 1,41 =6.40, P=0.015, Fig. 3). Telome e leng h o ledgling magpies inc eased as he season p og essed (R=0.49, =2.83, N=27, P=0.009, Fig. 3), e en a e con olling o he e ec o elome e leng h a ha ching (laying da e: pa ial Fig. 1 Mean con idence in e als (95%) o elome e leng h (NRQ alues) o g ea spo ed cuckoo and magpie ha chlings and ledglings. Lines connec ing alues o he same species indica e elome e dynamics du ing he nes ing phase. Fig. 2 Rela ionships be ween elome e leng h a ha ching ime and laying da e o magpies (open ci cles, con inuous lines) and g ea spo ed cuckoos ( ull ci cles, do ed line). Values a e mean nes alues and lines a e eg ession lines. ª2015 EUROPEAN SOCIETY FOR EVOLUTIONARY BIOLOGY. J. EVOL. BIOL. doi: 10.1111/jeb.12680 JOURNAL OF EVOLUTIONARY BIOLOGY ª2015 EUROPEAN SOCIETY FOR EVOLUTIONARY BIOLOGY 4JUAN J. SOLER ET AL. R=0.50, P=0.021; elome e leng h a ha ching: pa ial R=0.02, P=0. 93). This end was no de ec ed in cuck- oos o which la e b oods had nonsigni ican smalle elome es han hose o ea ly b oods (R=0.30, =1.25, N=18, P=0.23, Fig. 3) e en a e con olling o he e ec o elome e leng h a ha ching (laying da e: pa ial R=0.01, P=0.96; elome e leng h a ha ching: pa ial R=0.27, P=0.36). The abo e epo ed associa ions be ween laying da e and elome e leng h o ha chlings and ledglings o g ea spo ed cuckoos and magpies sugges ha he associa ions be ween elome e a i ion and laying da e should also di e o magpie and g ea spo ed cuckoo nes lings (ANCOVA, in e ac ion be ween species iden i y and laying da e, F 1,36 =10.15, P=0.003, Fig. 4). Telome e a i ion du ing he nes ing phase was nega- i ely associa ed wi h laying da e o magpie nes lings (R=0.53, =3.10, N=26, P=0.005, Fig. 4) e en a e con olling o he e ec o elome e leng h a ha ching (laying da e: pa ial R=0.46, P=0.021; elome e leng h a ha ching: pa ial R=0.95, P<0.0001). The posi i e associa ion de ec ed o g ea spo ed cuckoos (R=0.44, =1.69, N=14, P=0.12, Fig. 4) was a om s a is ical signi icance a e con ol- ling o elome e leng h a ha ching (laying da e: pa ial R=0.01, P=0.96; elome e leng h a ha ching: pa - ial R=0.69, P=0.008). Discussion Ou main inding is ha elome e leng h o magpie nes lings is d as ically educed du ing de elopmen a he nes , whe eas no changes occu in cuckoo nes lings. This sugges s ha magpies a e su e ing highe le els o s ess han cuckoos o ha cuckoos can deal wi h such s ess be e han magpies. Mo eo e , we ound in e - speci ic di e ences in he associa ion be ween laying da e and elome e leng h and sho ening. The s onge associa ions we e de ec ed in magpies o which la e ha chlings had he sho e elome es, whe eas elome e a i ion was s onge o magpie nes lings o ea ly b oods. Below we discuss hese esul s in he con ex s o in e speci ic di e ences in de elopmen al s a egies o g ea spo ed cuckoos and magpies ha may a ec elome e leng h and en i onmen al in luences on he dynamics o elome e change. In e speci ic di e ences in elome e leng h along he nes ling pe iod When g ea spo ed cuckoos success ully pa asi ize mag- pie nes s be o e he onse o incuba ion, he accele a ed emb yonic de elopmen al a es o pa asi es ela i e o hose o hei hos s con e pa asi ic o sp ing wi h huge ad an ages when compe ing o ood wi h hos nes - lings (Sole & Sole , 1991). In hese pa asi ized nes s, hos nes lings ha ch i e o six days a e cuckoos ha ch, a a ime when pa asi ic os e siblings weigh mo e han 60 g, which is en imes mo e han he weigh o magpie ha chlings (J.J. Sole , unpublished in o ma ion). Because magpie adul s p e e o eed he la ges nes ling in he nes (Sole e al., 1995), magpie o sp ing has no op ion o su i e in hese nes s whe e success ul pa asi ism occu s be o e incuba ion s a ed (Sole e al., 1998). Thus, i is likely ha a e paying he cos s o accele a ed a e o cell di ision du ing hei sho emb yonic s age, cuckoos will enjoy a peace ul nes ling pe iod wi h no appa en esou ce limi a ion o g owing (Sole & Sole , 2000). In con as , magpie nes - lings should always compe e o ood wi h siblings and migh he e o e su e inc eased cos s in e ms o elom- e e a i ion (see In oduc ion). In ag eemen wi h his Fig. 3 Rela ionships be ween elome e leng h a ledging ime and laying da e o magpies (open ci cles, con inuous lines) and g ea spo ed cuckoos ( ull ci cles, do ed line). Values a e mean nes alues and lines a e eg ession lines. Fig. 4 Rela ionships be ween elome e a i ion du ing de elopmen in magpies (open ci cles, con inuous lines) and g ea spo ed cuckoos ( ull ci cles, do ed line) and laying da e. Values a e mean nes alues and lines a e eg ession lines. ª2015 EUROPEAN SOCIETY FOR EVOLUTIONARY BIOLOGY. J. EVOL. BIOL. doi: 10.1111/jeb.12680 JOURNAL OF EVOLUTIONARY BIOLOGY ª2015 EUROPEAN SOCIETY FOR EVOLUTIONARY BIOLOGY B ood pa asi ism and elome e dynamics 5 scena io, we ound ha elome es o magpies, bu no o g ea spo ed cuckoos, dec eased signi ican ly o e he nes ing pe iod, which is in acco dance wi h di e - en ial cos s associa ed wi h in ense in ab ood compe i- ion in e ms o elome e a i ion de ec ed in some o he species (Salomons e al., 2009b; Ne le e al., 2013). Pa icula ly in iguing is he lack o e idence o con- sis en elome e a i ion o cuckoo nes lings o e he nes ing pe iod. E en mo e when conside ing ha o one- hi d o he s udied nes s wi h g ea spo ed cuck- oos, elome e leng h inc eased a he han dec eased (Fig. 4), al hough some o hese cases may be explained by measu emen e o s. Telome e enla gemen o e he nes ling pe iod has been p e iously de ec ed in s a - lings, bu because elome ase ac i i y is conside ed o be down- egula ed in ju eniles as a umou -p o ec ing mechanism (W igh & Shay, 2001), and i was only de ec ed in wo indi iduals, hese we e conside ed as ou lie s due o measu emen e o s (Ne le e al., 2013). Howe e , con a y o he idea ha elome ase ac i i y is mainly supp essed in mos soma ic issues o de elop- ing animals, Haussmann e al. (2007) de ec ed ha i is main ained h oughou he li espan o bi ds, including he ha chling age class. Thus, i is possible ha elom- e ase ac i i y was especially high in g ea spo ed cuckoo nes lings, coun e ac ing degene a i e e ec s o he apid g ow h expe ienced du ing he nes ling s age, possibly e en allowing some eco e y o elome e leng h a e hei ex emely speedy g ow h du ing he emb yonic phase (and he p esumably e y high elom- e e a i ion a e du ing his phase, see abo e). How- e e , i is also possible ha he in e speci ic di e ences in elome e ajec o ies we e due o di e ences in he capaci y o con ol oxida i e s ess (i.e. highe access o an ioxidan s gi en a highe access o ood o mo e in ense an ioxidan enzyma ic ac i i y) (Bad as e al., 2015). Finally, because ledgling cuckoos we e sampled when hey we e wo days younge han magpies, di - e ence in age migh explain he in e speci ic di e - ences de ec ed in elome e a i ion. This las possibili y is, howe e , unlikely as cuckoos de elop as e han magpies and nes lings o bo h species a e a simila de elopmen al s age a he age o sampling (Sole & Sole , 1991). Telome e leng h and sho ening, and laying da e Laying da e is one o he mos employed a iables e lec ing e i o y quali y and a ailabili y o esou ces and/o pa en al quali y (B inkho e al., 1993; Mo eno, 1998; De Ne e e al., 2004; Ve huls & Nilsson, 2008). Inc eased nes ling compe i ion educes esou ce a ail- abili y o g owing nes lings, and de e io a ion o qual- i y o pa en al ca e is pos -na al s esso s ypically associa ed wi h laying da e (Pe ins, 1970; B inkho e al., 1993). The e o e, an inc ease in endogenous glu- coco icoids is p edic ed in nes lings om la e b eeding a emp s (Bane jee e al., 2012), which consequen ly should a ec elome e leng h dynamics (Monaghan, 2014). In acco dance wi h his scena io, we ound ha la e magpie ha chlings had sho e elome e leng h han ea ly ha chlings. Howe e , some o ou esul s we e no in acco dance wi h his hypo hesis as elome e leng h o g ea spo ed cuckoo ha chlings was posi i ely associa ed wi h laying da e, and elome e a i ion expe ienced by magpie nes lings dec eased o e he b eeding season; a endency no de ec ed in cuckoos. The de ec ed associa- ions be ween laying da e and ei he elome e leng h o elome e a i ion did no depend on elome e leng h a ha ching, and he e o e, hese associa ions canno be explained by he commonly desc ibed highe elome e a i ion in indi iduals wi h ini ially longe elome es (Ma cand e al., 1999; Ka lsede e al., 2002; Hall e al., 2004). Thus, in e speci ic di e ences in he associa ion be ween elome e leng h and laying da e s ongly sug- ges ha ela ed en i onmen al cha ac e is ics do in lu- ence elome e dynamics o magpie and o g ea spo ed cuckoo nes lings di e en ly. I is di icul , howe e , o explain con adic o y esul s o he p oposed scena io, and we can only specula e abou possible easons. I is, o ins ance, possible ha he nega i e associa ion be ween elome e leng h o magpie ha chlings and lay- ing da e was due o a ia ion in esou ce a ailabili y o laying emales (Pe ins, 1970; D en , 2006). La e magpie nes lings ha e poo e immune sys em (So ci e al., 1997) and lowe de elopmen al a e han ea ly nes - lings (De Ne e e al., 2004), which may hus explain he de ec ed nega i e co ela ion be ween laying da e and elome e a i ion o magpies du ing de elopmen . How- e e , his possibili y p edic s a posi i e ela ionship be ween elome e a i ion and laying da e ha we did no ind. Ano he possibili y explaining he de ec ed el- a i ely la ge elome e a i ion a es o ea ly magpie ha chlings is ha la e ha chlings expe ience a ela i ely low wi hin-b ood compe i ion. Howe e , esou ce a ail- abili y dec eases as he season p og esses in magpie nes s (De Ne e e al., 2004), and i is likely nega i ely ela ed wi h le el o wi hin-b ood compe i ion. Addi- ionally, elome e leng h has a ma e nal and a he edi- a y componen in bi ds (Monaghan, 2014; Asgha e al., 2015b), and consequen ly, ela ionships be ween ha chling elome e leng h and laying da e may e lec an unde lying ela ionship be ween he gene ic basis o elome e leng h o pa en s (mainly emales) and laying da e. Taken oge he , all hese esul s o he i s ime poin ou a ela ionship be ween en i onmen al condi- ions (laying da e) and elome e leng h and sho ening ha a y be ween species expe iencing simila ecologi- cal condi ions du ing g ow h. As elome e leng h and elome e sho ening a e a iables e lec ing su i al p ospec and cos s associa ed wi h s ess esponses ª2015 EUROPEAN SOCIETY FOR EVOLUTIONARY BIOLOGY. J. EVOL. BIOL. doi: 10.1111/jeb.12680 JOURNAL OF EVOLUTIONARY BIOLOGY ª2015 EUROPEAN SOCIETY FOR EVOLUTIONARY BIOLOGY 6JUAN J. SOLER ET AL. (Asgha e al., 2015a; Ne le e al., 2015), hese esul s may sugges ha species a ying in de elopmen al s a egies (i.e. nes ling g ow h and/o emb yonic g ow h a es) also a y in su e ed cos s in e ms o elome e sho ening and/o dynamics. The e o e, ou esul s i expec a ions acco ding o in e speci ic di e ences in li e- s yle and li e his o y. Explo ing possible causes o he de ec ed in e speci ic di e ences and associa ions may help o unde s and he e olu iona y ela ionship be ween elome e dynamics and li e-his o y ai s o ani- mals. Acknowledgmen s We hank Olga Co ona Fo e o, F ancisco Mi anda and Olaya Ga c ıa o echnical assis ance wi h DNA ex ac- ion om blood samples and ampli ica ion o elome e leng h es ima ion. Funding was p o ided by he Span- ish Minis e io de Economia y compe i i idad and Eu o- pean Funds (FEDER) (CGL2013-48193-C3-1-P, CGL2012-30759) and Jun a de Andaluc ıa (RNM 340, RNM7038). JMP is suppo ed by a Juan de la Cie a con ac . Blood sampling and nes isi ing we e done ollowing e hical s anda ds and unde he pe mission o Jun a de Andaluc ıa, En i onmen al Managemen Agency, which au ho ized ield p o ocol (Re .: SGMN/ GyB/JMIF). S a emen o au ho ship JJS and JF designed he s udy wi h conside able assis- ance om JMP and CRC. CRC pe o med all molecula analyses wi h conside able assis ance JMP. JJS, MRR and GT pe o med mos o he ield wo k wi h assis- ance by CRC. JJS pe o med all he s a is ical analyses and w o e he manusc ip wi h subs an ial con ibu ion om all au ho s Re e ence Asgha , M., Hasselquis , D., Hansson, B., Zeh indjie , P., Wes- e dahl, H. & Bensch, S. 2015a. Hidden cos s o in ec ion: ch onic mala ia accele a es elome e deg ada ion and senes- cence in wild bi ds. Science 347: 436–438. Asgha , M., Bensch, S., Ta ka, M., Hansson, B. & Hasselquis , D. 2015b. Ma e nal and gene ic ac o s de e mine ea ly li e elome e leng h. P oc. R. Soc. Lond. B 282: 20142263. Bad as, E.P., Ma  ınez, J., Ri e o de Aguila Cacha ei o, J., Mi- anda, F., Figue ola, J. & Me ino, S. 2015. Ageing and ep o- duc ion: an ioxidan supplemen a ion alle ia es elome e loss in wild bi ds. J. E ol. Biol. 28: 896–905. Bane jee, S.B., A e be y, A.S., Fe gus, D.J. & Adkins-Regan, E. 2012. Dep i a ion o ma e nal ca e has long-las ing conse- quences o he hypo halamic-pi ui a y-ad enal axis o zeb a inches. P oc. R. Soc. Lond. B 279: 759–766. Bee y, A.K., Lin, J., Biddle, J.S., F ancis, D.D., Blackbu n, E.H. & Epel, E.S. 2012. Ch onic s ess ele a es elome ase ac i i y in a s. Biol. Le . 8: 1063–1066. Blackbu n, E.H. 1991. S uc u e and unc ion o elome es. Na- u e 350: 569–573. Blanco, G., F ias, O., Ma inez, J., Lemus, J.A., Me ino, R. & Jimenez, B. 2006. Sex and ank in compe i i e b ood hie a - chies in luence s ess le els in nes lings o a sexually dimo - phic bi d. Biol. J. Linn. Soc. 88: 383–390. Boonekamp, J.J., Mulde , G.A., Salomons, H.M., Dijks a, C. & Ve huls , S. 2014. Nes ling elome e sho ening, bu no elome e leng h, e lec s de elopmen al s ess and p edic s su i al in wild bi ds. P oc. R. Soc. Lond. B 281: 20133287. B inkho , M.W.G., Ca  e, A.J., Hage, F.J. & Ve huls , S. 1993. Timing o ep oduc ion and ledging success in he coo Fulica a a: e idence o a causal ela ionship. J. Anim. Ecol. 62: 577–587. Cap ioli, M., Romano, M., Romano, A., Rubolini, D., Mo a, R., Folini, M. e al. 2013. Nes ling elome e leng h does no p edic longe i y, bu co a ies wi h adul body size in wild ba n swallows. Biol. Le . 9: 0340. C iscuolo, F., Bize, P., Nasi , L., Me cal e, N.B., Foo e, C.G., G i i hs, K. e al. 2009. Real- ime quan i a i e PCR assay o measu emen o a ian elome es. J. A ian Biol. 40: 342–347. De Ne e, L., Sole , J.J., Sole , M. & P e ez-Con e as, T. 2004. Di e en ial ma e nal in es men coun e ac s o la e b eed- ing in magpies Pica pica: an expe imen al s udy. J. A ian Biol. 35: 237–245. D en , R.H. 2006. The iming o bi ds’ b eeding seasons: The Pe ins hypo hesis e isi ed especially o mig an s. A dea 94: 305–322. Fe agu i, M., Ma inez-de la Puen e, J., Ruiz, S., So igue , R. & Figue ola, J. 2013. On he s udy o he ansmission ne - wo ks o blood pa asi es om SW Spain: di e si y o a ian haemospo idians in he bi ing midge Culicoides ci cumsc ip- us and wild bi ds. Pa asi . Vec o s 6: 208. Foo e, C.G., Gaul , E.A., Nasi , L. & Monaghan, P. 2011. Telome e dynamics in ela ion o ea ly g ow h condi ions in he wild in he lesse black-backed gull. J. Zool. (Lond.) 283: 203–209. Fo sy h, N.R., W igh , W.E. & Shay, J.W. 2002. Telome ase and di e en ia ion in mul icellula o ganisms: u n i o , u n i on, and u n i o again. Di e en ia ion 69: 188–197. Hall, M.E., Nasi , L., Daun , F., Gaul , E.A., C oxall, J.P., Wanless, S. e al. 2004. Telome e loss in ela ion o age and ea ly en i on- men in long-li ed bi ds. P oc. R. Soc. Lond. B 271: 1571–1576. Haussmann, M.F., Winkle , D.W., Hun ing on, C.E., Nisbe , I.C.T. & Vleck, C.M. 2007. Telome ase ac i i y is main ained h oughou he li espan o long-li ed bi ds. Exp. Ge on ol. 42: 610–618. Heidinge , B.J., Bloun , J.D., Bone , W., G i i hs, K., Me cal e, N.B. & Monaghan, P. 2012. Telome e leng h in ea ly li e p edic s li espan. P oc. Na l. Acad. Sci. USA 109: 1743–1748. Hellemans, J., Mo ie , G., De Paepe, A., Speleman, F. & Van- desompele, J. 2007. qBase ela i e quan i ica ion amewo k and so wa e o managemen and au oma ed analysis o eal- ime quan i a i e PCR da a. Genome Biol. 8:1–14. He bo n, K.A., Heidinge , B.J., Bone , W., Nogue a, J.C., Adam, A., Daun , F. e al. 2014. S ess exposu e in ea ly pos -na al li e educes elome e leng h: an expe imen al demons a ion in a long-li ed seabi d. P oc. R. Soc. Lond. B 281: 3151. Ka lsede , J., Smogo zewska, A. & de Lange, T. 2002. Senes- cence induced by al e ed elome e s a e, no elome e loss. Science 295: 2446–2449. ª2015 EUROPEAN SOCIETY FOR EVOLUTIONARY BIOLOGY. J. EVOL. BIOL. doi: 10.1111/jeb.12680 JOURNAL OF EVOLUTIONARY BIOLOGY ª2015 EUROPEAN SOCIETY FOR EVOLUTIONARY BIOLOGY B ood pa asi ism and elome e dynamics 7 Ko schal, A., Ilmonen, P. & Penn, D.J. 2007. S ess impac s elome e dynamics. Biol. Le . 3: 128–130. Ma cand, S., B e e , V. & Gilson, E. 1999. P og essi e cis-inhi- bi ion o elome ase upon elome e elonga ion. EMBO J. 18: 3509–3519. Ma  ınez-Padilla, J., Ma inez, J., D a ila, J.A., Me ino, S., Mo eno, J. & Mill an, J. 2004. Wi hin-b ood size di e ences, sex and pa asi es de e mine blood s ess p o ein le els in Eu asian Kes el nes lings. Func . Ecol. 18: 426–434. Ma  ın-G al ez, D., P e ez-Con e as, T., Sole , M. & Sole , J.J. 2011. Bene i s associa ed wi h escala ed begging beha iou o black-billed magpie nes lings o e compensa e he associ- a ed ene ge ic cos s. J. Exp. Biol. 214: 1463–1472. Mock, D.W. & Pa ke , G.A. 1997. The E olu ion o Sibling Ri - al y. Ox o d Uni e si y P ess, Ox o d. Monaghan, P. 2008. Re iew. Ea ly g ow h condi ions, pheno- ypic de elopmen and en i onmen al change. Phil. T ans. R. Soc. Lond. B 363: 1635–1645. Monaghan, P. 2014. O ganismal s ess, elome es and li e his- o ies. J. Exp. Biol. 217:57–66. Monaghan, P. & Haussmann, M.F. 2006. Do elome e dynamics link li es yle and li espan?. T ends Ecol. E ol. 21: 47–53. Mo eno, J. 1998. The de e mina ion o seasonal declines in b eeding success in seabi ds. E olog ıa 6:17–31. Ne le, D., Monaghan, P., Bone , W., Gillespie, R. & Ba eson, M. 2013. Bo om o he heap: ha ing hea ie compe i o s accele a es ea ly-li e elome e loss in he Eu opean s a ling, S u nus ulga is.PLoS ONE 8: e83617. Ne le, D., Monaghan, P., Gillespie, R., B ilo , B., Bed o d, T. & Ba eson, M. 2015. An expe imen al demons a ion ha ea ly-li e compe i i e disad an age accele a es elome e loss. P oc. R. Soc. Lond. B 282: 20141610. Nussey, D.H., Bai d, D., Ba e , E., Bone , W., Fai lie, J., Gemmell, N. e al. 2014. Measu ing elome e leng h and elome e dynamics in e olu iona y biology and ecology. Me hods Ecol. E ol. 5: 299–310. Pe ins, C.M. 1970. Timing o bi ds b eeding seasons. The Ibis 112: 242–255. Reiche , S., S ie , A., Zahn, S., A i e, M., Bize, P., Massemin, S. e al. 2014. Inc eased b ood size leads o pe sis en e oded elome es. F on . Ecol. E ol. 2: doi:10.3389/ e o.2014.00009. Rickle s, R.E. & Finch, C.E. 1995. Aging. A Na u al His o y. Sci- en i ic Ame ican Lib a y, New Yo k. Sahin, E. & DePinho, R.A. 2010. Linking unc ional decline o elome es, mi ochond ia and s em cells du ing ageing. Na u e 464: 520–528. Salomons, H.M., Mulde , E. & Ve huls , S. 2009a. Telome e leng h a ia ion and sex dependen en i onmen al sensi i - i y du ing ea ly de elopmen in a ee-li ing co id. In: Figh ing o Fi ness: Telome es, Oxida i e S ess and Li e His o y T ade-O s in a Colonial Co id (H.M. Salomons, ed.), pp. 53– 62. PhD hesis, Uni e si y o G oningen, G oningen. Salomons, H.M., Mulde , G.A., an de Zande, L., Haussmann, M.F., Linskens, M.H.K. & Ve huls , S. 2009b. Telome e sho ening and su i al in ee-li ing co ids. P oc. R. Soc. Lond. B 276: 3157–3165. Sole , M. & De Ne e, L. 2013. B ood ma e e ic ion o b ood ma e accep ance by b ood pa asi ic nes lings? An expe imen- al s udy wi h he non-e ic o g ea spo ed cuckoo and i s magpie hos . Beha . Ecol. Sociobiol. 67: 601–607. Sole , M. & Sole , J.J. 1991. G ow h and de elopmen o g ea spo ed cuckoos and hei magpie hos . Condo 93:49–54. Sole , J.J. & Sole , M. 2000. B ood-pa asi e in e ac ions be ween g ea spo ed cuckoos and magpies: a model sys em o s udying coe olu iona y ela ionships. Oecologia 125: 309–320. Sole , M., Ma  ınez, J.G., Sole , J.J. & Mølle , A.P. 1995. P e - e en ial alloca ion o ood by magpie Pica pica o g ea spo - ed cuckoo Clama o glanda ius chicks. Beha . Ecol. Sociobiol. 37:7–13. Sole , M., Sole , J.J. & Ma  ınez, J.G. 1998. Du a ion o sym- pa y and coe olu ion be ween he g ea spo ed cuckoo (Clama o glanda ius) and i s p ima y hos , he magpie (Pica pica). In: Pa asi ic Bi ds and Thei Hos s, S udies in Coe olu ion (S.I. Ro hs ein, S.K. Robinson, eds), pp. 113–128. Ox o d Uni e si y P ess, Ox o d. Sole , J.J., Mølle , A.P., Sole , M. & Ma  ınez, J.G. 1999. In e - ac ions be ween a b ood pa asi e and i s hos in ela ion o pa asi ism and immune de ence. E ol. Ecol. Res. 1: 189–210. Sole , J.J., Ma  ınez, J.G., Sole , M. & Mølle , A.P. 2001. Coe olu iona y in e ac ions in a hos -pa asi e sys em. Ecol. Le . 4: 470–476. Sole , J.J., Ma in-Gal ez, D., De Ne e, L. & Sole , M. 2013. B ood pa asi ism co ela es wi h he s eng h o spa ial au o- co ela ion o li e his o y and de ensi e ai s in Magpies. Ecology 94: 1338–1346. So ci, G., Sole , J.J. & Mølle , A.P. 1997. Reduced immuno- compe ence o nes lings in eplacemen clu ches o he Eu opean magpie (Pica pica). P oc. R. Soc. Lond. B 264: 1593– 1598. S a so Inc. 2011. STATISTICA (Da a Analysis So wa e Sys em), Ve sion 10. www.s a so .com. S ensson, E. 1995. A ian ep oduc i e iming: when should pa en s be p uden ? Anim. Beha . 49: 1569–1575. Ve huls , S. & Nilsson, J.A. 2008. The iming o bi ds’ b eeding seasons: a e iew o expe imen s ha manipula ed iming o b eeding. Phil. T ans. R. Soc. Lond. B 363: 399–410. W igh , W.E. & Shay, J.W. 2001. Cellula senescence as a umo -p o ec ion mechanism: he essen ial ole o coun ing. Cu . Opin. Gene . De . 11:98–103. Da a deposi ed a D yad: doi: 10.5061 d yad.6n 62. Recei ed 19 Ap il 2015; e ised 14 June 2015; accep ed 16 June 2015 ª2015 EUROPEAN SOCIETY FOR EVOLUTIONARY BIOLOGY. J. EVOL. BIOL. doi: 10.1111/jeb.12680 JOURNAL OF EVOLUTIONARY BIOLOGY ª2015 EUROPEAN SOCIETY FOR EVOLUTIONARY BIOLOGY 8JUAN J. SOLER ET AL.