Geographical Variation in Egg Mass and Egg Content in a Passerine Bird

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Geog aphical Va ia ion in Egg Mass and Egg Con en in a Passe ine Bi d Ruuskanen, Su i; Sii a i, Heli; Ee a, Tapio; Belskii, Eugen; Jä inen, An e o; Ke imo , An a ; K ams, Ind ikis; Mo eno, Juan; Mo osino o, Chia a; Mänd, Rai o; Mös l, E ich; O ell, Ma kku; Q a ns öm, Anna; Salminen, Juha-Pekka; Sla e , F ed; Tilga , Vallo; Visse , Ma cel E.; Winkel, Wol gang; Zang, He wig; Laaksonen, Toni Ruuskanen, S., Sii a i, H., Ee a, T., Belskii, E., Jä inen, A., e al. (2011) Geog aphical Va ia ion in Egg Mass and Egg Con en in a Passe ine Bi d. PLoS ONE 6(11): e25360. doi:10.1371/jou nal.pone.0025360 2011 Geog aphical Va ia ion in Egg Mass and Egg Con en in a Passe ine Bi d Su i Ruuskanen 1 *, Heli Sii a i 2 , Tapio Ee a 1 , Eugen Belskii 3 , An e o Ja ¨ inen 4 , An a Ke imo 5 , Ind ikis K ams 6 , Juan Mo eno 7 , Chia a Mo osino o 1 , Rai o Ma ¨nd 8 , E ich Mo ¨s l 9 , Ma kku O ell 10 , Anna Q a ns o ¨m 11 , Juha-Pekka Salminen 12 , F ed Sla e 13 , Vallo Tilga 8 , Ma cel E. Visse 14 , Wol gang Winkel 15 , He wig Zang 16 , Toni Laaksonen 1,17 1Sec ion o Ecology, Depa men o Biology, Uni e si y o Tu ku, Tu ku, Finland, 2Depa men o Biological and En i onmen al Sciences, Uni e si y o Jy a ¨skyla ¨, Jy a ¨skyla ¨, Finland, 3Ins i u e o Plan and Animal Ecology, Russian Academy o Sciences, Eka e inbu g, Russia, 4Facul y o Biological and En i onmen al Sciences, Uni e si y o Helsinki, Helsinki, Finland, 5Z enigo od Biological S a ion o Moscow S a e Uni e si y, Moscow, Russia, 6Ins i u e o Sys ema ic Biology, Dauga pils Uni e si y, Dauga pils, La ia, 7Depa amen o de Ecologıa E olu i a, Museo Nacional de Ciencias Na u ales-CSIC, Mad id, Spain, 8Depa men o Zoology, Uni e si y o Ta u, Ta u, Es onia, 9Uni e si y o Ve e ina y Medicine, Vienna, Aus ia, 10 Depa men o Biology, Uni e si y o Oulu, Oulu, Finland, 11 Depa men o Animal Ecology, Uni e si y o Uppsala, Uppsala, Sweden, 12 Labo a o y o O ganic Chemis y and Chemical Biology, Uni e si y o Tu ku, Tu ku, Finland, 13 School o Biosciences, Ca di Uni e si y, Ca di , Uni ed Kingdom, 14 Ne he lands Ins i u e o Ecology, Wageningen, The Ne he lands, 15 Ins i u e o A ian Resea ch ‘Vogelwa e Helgoland’, Wilhelmsha en, Ge many, 16 Gosla , Ge many, 17 Finnish Museum o Na u al His o y, Uni e si y o Helsinki, Helsinki, Finland Abs ac Rep oduc i e, pheno ypic and li e-his o y ai s in many animal and plan axa show geog aphic a ia ion, indica ing spa ial a ia ion in selec ion egimes. Ma e nal deposi ion o a ian eggs, such as ho mones, an ibodies and an ioxidan s, c i ically a ec de elopmen o he o sp ing, wi h long-las ing e ec s on he pheno ype and i ness. Li le is howe e known abou la ge-scale geog aphical pa e ns o a ia ion in ma e nal deposi ion o eggs. We s udied geog aphical a ia ion in egg componen s o a passe ine bi d, he pied lyca che (Ficedula hypoleuca), by collec ing samples om 16 popula ions and measu ing egg and yolk mass, albumen lysozyme ac i i y, yolk immunoglobulins, yolk and ogens and yolk o al ca o enoids. We ound signi ican a ia ion among popula ions in mos egg componen s, bu ca. 90% o he a ia ion was among indi iduals wi hin popula ions. Popula ion howe e explained 40% o he a ia ion in ca o enoid le els. In con as o ou hypo hesis, we ound geog aphical ends only in ca o enoids, bu no in any o he o he egg componen s. Ou esul s hus sugges high wi hin-popula ion a ia ion and lea e li le scope o local adap a ion and gene ic di e en ia ion in deposi ion o di e en egg componen s. The ole o hese ma e nally-de i ed esou ces in e olu iona y change should be u he in es iga ed. Ci a ion: Ruuskanen S, Sii a i H, Ee a T, Belskii E, Ja ¨ inen A, e al. (2011) Geog aphical Va ia ion in Egg Mass and Egg Con en in a Passe ine Bi d. PLoS ONE 6(11): e25360. doi:10.1371/jou nal.pone.0025360 Edi o : Ke in McG aw, A izona S a e Uni e si y, Uni ed S a es o Ame ica Recei ed Ap il 15, 2011; Accep ed Sep embe 1, 2011; Published No embe 14, 2011 Copy igh : ß2011 Ruuskanen e al. This is an open-access a icle dis ibu ed unde he e ms o he C ea i e Commons A ibu ion License, which pe mi s un es ic ed use, dis ibu ion, and ep oduc ion in any medium, p o ided he o iginal au ho and sou ce a e c edi ed. Funding: This s udy was inancially suppo ed by Tu ku Uni e si y Founda ion, Finnish Cul u al Founda ion 381 (g an s o S.R.) and Emil Aal onen Founda ion (a g an o T.L.). The unde s had no ole in s udy design, da a collec ion and analysis, decision o publish, o p epa a ion o he manusc ip . Compe ing In e es s: The au ho s ha e decla ed ha no compe ing in e es s exis . * E-mail: su i. uuskanen@u u. i In oduc ion Pheno ypes, li e-his o ies and ep oduc i e s a egies in many animal and plan axa show geog aphic a ia ion, indica ing spa ial a ia ion in selec ion egimes [1–6]. In pa icula , ep oduc i e in es men , in he o m o clu ch o egg size, shows la i udinal ends in e - and in a-speci ically ac oss axonomic g oups [7–10] and is pa icula ly well s udied in bi ds [11,12]. A numbe o hypo heses ha e been pu o wa d o explain he la i udinal a ia ion in a ian ep oduc i e in es men (clu ch and egg size), including ene gy, nu ien o ime limi a ions o a ia ion in p eda ion p essu e [12–17]. In gene al, ep oduc i e in es men (e.g. egg size) is subjec o se e al selec ion p essu es: pa en - o sp ing con lic o e pe -o sp ing in es men , ade-o be ween egg size and numbe , and compe ing demands o esou ces in he pa en s [18]. Thus i selec ion op imizes o sp ing i ness, dec easing en i onmen al p oduc i i y wi h la i ude may be expec ed o lead o inc easing in es men in egg quali y wi h la i ude, o inc ease o sp ing su i al [18–20]. Al e na i ely, highe adul main enance cos s in poo en i onmen al condi ions may lead o educed in es men in eggs [18], which could gene a e a dec easing la i udinal end in ep oduc i e in es men . In addi ion o egg size, ma e nal in es men in o sp ing quali y in he o m o di e en egg componen s such as lipids, immune ac o s, ho mones and an ioxidan s c i ically in luences o sp ing de elopmen and su i al in many axa [18,21–27]. Ma e nally- de i ed immunoglobulins p o ide he p ima y o m o humo al immune de ence o he o sp ing, as unde de eloped young canno syn hesize hem [28]. Lysozyme enzyme des oys cell walls o bac e ia and i is hus a majo componen o he an ibac e ial immuni y o he egg [29]. Yolk and ogens can a ec o sp ing de elopmen and pheno ype in many ways, o example g ow h, immuni y, beha iou and plumage ai s [25]. Ca o enoids a e an ioxidan s ha educe lipid pe oxida ion in he emb yo, and hey can also enhance immune unc ion [30]. Ca o enoid le els a e mainly de e mined by hei a ailabili y in he mo he ’s die , PLoS ONE | www.plosone.o g 1 No embe 2011 | Volume 6 | Issue 11 | e25360 because hey canno be syn hesized by bi ds o s o ed o a long ime [30]. Deposi ion o se e al egg componen s is known o be a ec ed by en i onmen al o social condi ions (e.g. ood a ailabili y, pa asi e load o quali y o ma es) wi hin popula ions [25,30–34]. To ou knowledge only e y ew s udies ha e es ima ed la ge-scale geog aphical a ia ion among popula ions in egg componen s (o any ma e nal e ec s) in any species (wi h he excep ion o egg size). Mos o he exis ing s udies in bi ds ha e compa ed deposi ion in o eggs in wo con as ing en i onmen s [35–40], bu hese esul s sugges ha popula ions could di e in se e al ma e nally-de i ed egg componen s (e.g. yolk ca o enoids and and ogens). Ma e nal e ec s, ia ma e nal beha io o esou ce alloca ion, ha e been sugges ed o play an impo an ole in ai e olu ion and e en popula ion di e en ia ion [41–44]. S udying geog aph- ical a ia ion in ma e nal deposi ion o eggs may be seen as he i s s ep o e eal i s e olu iona y po en ial. As wi h o he li e- his o y ai s, spa ial a ia ion in adap i e bene i s o ma e nal esou ce alloca ion may lead o among-popula ion a ia ion in egg quali y [6]. Gi en ha deposi ion o se e al egg componen s a ec s o sp ing i ness and is he i able [25,27,45–47], selec ion on egg composi ion may lead o mic oe olu ion in hese ai s. Al e na i ely, among-popula ion a ia ion in deposi ion o eggs could be due o pheno ypic plas ici y (ei he due o esou ce limi a ion o adap i e esou ce alloca ion), which may e en cons ain gene ic di e en ia ion [48]. We s udied geog aphical a ia ion in egg componen s o he pied lyca che (Ficedula hypoleuca) by collec ing egg samples om 16 popula ions all o e Eu ope and analysing a ia ion in egg and yolk mass, albumen lysozyme enzyme ac i i y, yolk immunoglob- ulin, yolk and ogen ( es os e one and and os enedione) and yolk ca o enoid concen a ions. The s udy species shows geog aphical a ia ion in ep oduc i e ai s: Timing o b eeding ge s la e and clu ch size dec eases owa ds he no h, whe e b eeding seasons a e sho e and mo e unp edic able, empe a u es a e lowe and ood a ailabili y du ing egg-laying may be mo e limi ed (as egg laying begins a ea lie ambien phenology) [15,49–52]. Egg size has been ound o inc ease (linea o quad a ic ends) owa ds he no h in o he Eu opean passe ines [53,54], bu ends in he pied lyca che a e unclea [55]. Fo example, in es men in la ge eggs may be selec ed in no he n la i udes because o lowe ha ching ailu e o la ge eggs in cold empe a u es [56]. Wi hin-popula ion a ia ion in egg componen s in ela ion o en i onmen al a iables in he pied lyca che and i s sis e species, he colla ed lyca che (Ficedula albicollis) has been e ealed in he ecen yea s: yolk ca o enoid and immunoglobulin le els ha e been ound o a y in ela ion wi h laying o de [57,58], iming o b eeding [59], emale condi ion [57,59] and ca e pilla a ailabili y (ca o enoids) [58,60]. Fu he mo e, among-clu ch a ia ion in yolk and ogen le els seems o be associa ed wi h en i onmen al ac o s such as iming o b eeding [61,62], ood supply (Laaksonen, T. unpublished), social s imula ion [59], emale cha ac e is ics such as condi ion [47,62], and male quali y [61,63]. Yolk and ogen alloca ion has also been shown o be epea able and he i able [47,62]. Howe e , no in o ma ion on he among-popula ion a ia ion in egg componen s in he s udy species exis s. Ou i s aim was o quan i y he ex en o among- and wi hin- popula ion a ia ion in egg mass and key egg componen s a a la ge geog aphical scale – a a ely s udied opic. We sugges ha la ge among-popula ion a ia ion may be an indica o o di e en ial selec ion and gene ic di e ences, bu i can also be induced by pheno ypic plas ici y in hese ai s. Low a ia ion among popula ions and la ge a ia ion wi hin popula ions, howe e , is likely o indica e li le scope o gene ic di e ences, po en ially high pheno ypic plas ici y, o sugges ha he e is no spa ial a ia ion in adap i e bene i s o ma e nal esou ce deposi ion o eggs. Al e na i ely, his may indica e ha emales a e cons ained in hei alloca ion, po en ially due o cos s o hemsel es o o sp ing [25]. Howe e , ou analysis is explo a o y and hus we canno sepa a e wi h ce ain y he cause o a ia ion among popula ions. Secondly, we s udied a ia ion in egg componen s in ela ion o geog aphical loca ion and habi a . We hypo hesized ha i ma e nal deposi ion o eggs imp o es o sp ing quali y, and i selec ion leads o inc easing o sp ing quali y in ma ginal en i onmen s [19,20], we should obse e a la i udinal inc ease in egg quali y in he s udy ange. Al e na i ely, i esou ces needed o sel -main enance (nu ien s o ene gy du ing egg laying) limi deposi ion o di e en egg componen s, an opposi e pa e n may eme ge. Howe e , i is no easy o de e mine which kind o deposi ion ep esen s imp o ed quali y o indi idual egg componen s, as wi h espec o some egg componen s, high le els may e en be de imen al (such as immunosupp ession by yolk and ogens) [25]. Di e en egg componen s may u he mo e be unde di e en cons ain s and selec i e o ces. We also in es iga ed whe he deposi ion o egg componen s is a ec ed by bio ic popula ion le el ac o s such as iming o b eeding o clu ch size and whe he deposi ion is associa ed wi h ep oduc i e success. Finally, we s udied he co- a ia ion be ween egg componen s, o examine he hypo hesis ha emales could simul aneously modi y deposi ion o se e al egg componen s. Fo example, high le els o yolk and ogens p omo e g ow h and may be immunosupp essi e, which may inc ease o sp ing need o an ioxidan s and ma e nal immune ac o s [64]. Al hough ou analyses a e explo a o y, we conside hese as an impo an i s s ep owa ds unde s anding a ia ion in esou ce alloca ion and he po en ial ole o ma e nal alloca ion o eggs in e olu iona y p ocesses. Me hods S udy species The pied lyca che is a small (12–13 g), mig a o y, insec i o- ous, hole-nes ing o es passe ine bi d [65]. I b eeds h oughou a la ge ange o e Eu ope and Wes e n Sibe ia (b eeding ange is illus a ed in Fig. 1) and win e s in wes e n A ica. Pied lyca che s a e single-b ooded and he modal clu ch size is i e o six eggs. The pied lyca che is an abundan species ha easily accep s a i icial nes boxes, and hus i is a common model species in s udies o a ian ecology and e olu ion. Field p o ocol Egg samples we e collec ed om 16 di e en nes -box s udy popula ions ac oss he b eeding ange o he pied lyca che du ing sp ing and summe 2007 (see Fig. 1). In each popula ion, nes - boxes we e checked a h ee day in e als o moni o he p og ess o nes ing. When eggs we e ound in he nes , hese we e ma ked and he nes was isi ed he ollowing days o collec he eshly laid hi d o ou h egg o each clu ch. Thus he collec ed eggs we e no incuba ed, and he e was no emb yo de elopmen . In each popula ion, egg sampling was conduc ed e enly h oughou he b eeding season i possible (lea ing ou e y i s and las nes s), o a oid bias in egg composi ion due o po en ial seasonal a ia ion in egg componen s. The egg componen s may a y sys ema ically wi h laying o de [25,33,57,58,62,66], bu he middle eggs should ep esen he a e age alues o he clu ch. Fo example, in a Finnish popula ion, he clu ch mean and ou h egg yolk and ogen le els we e highly co ela ed ( es os e one: s = 0.85, N = 24, p,0.001; and os enedione s = 0.78, N = 24, p,0.001). The Geog aphical Va ia ion in Egg Con en PLoS ONE | www.plosone.o g 2 No embe 2011 | Volume 6 | Issue 11 | e25360 posi ion o he egg in he laying sequence, laying da e and esh mass (,0.01 g, using s anda d scales, e.g. Pesola) we e eco ded. I mass could no be measu ed, he maximum leng h and wid h o he eggs we e measu ed (,0.01 mm, using digi al o manual calipe ) and he Hoy olume ( olume = leng h * b ead h 2 * 0.51) [67], which is highly co ela ed wi h he egg mass, was calcula ed. Hoy olume was u he con e ed o egg mass by mul iplying he olume by 1.06 [68]. Eggs we e he ea e s o ed a 220uC un il he labo a o y analyses. Nes s we e moni o ed h oughou he b eeding season, o eco d inal clu ch size and numbe o ha chlings and ledglings. Eggs we e collec ed in 16 popula ions, and om each popula ion, ca. 20 eggs we e acqui ed ( ange 4–32 eggs pe popula ion; see Table S1 o de ailed sample sizes). The sampling a ea co e s la ge pa s o he b eeding a ea o pied lyca che s in Eu ope. Loca ions o he sampling popula ions a e indica ed in Fig. 1 and Table S1. Da a om one popula ion (Es onia) we e collec ed om wo di e en habi a s (coni e ous and deciduous) o ming a mixed mosaic o habi a pa ches, which di e in some b eeding pa ame e s (e.g. laying da es, R. Ma¨nd, pe s. comm.). In he analyses hese wo we e conside ed as one popula ion, bu he habi a a iable was di e en o he wo habi a s. E hics s a emen Egg collec ion and all animal wo k was conduc ed unde ele an na ional and in e na ional guidelines and unde licenses om en i onmen al au ho i ies and e hical commi ees in each coun y (UK: Coun yside Council o Wales, license numbe OTH:SB:06:200; Sweden: Swedish Na ional Boa d o Labo a o- y Animals and he Bi d Ringing Cen e o he Swedish Museum o Na u al His o y, license numbe Dn 33-07; Spain: Conseje ı ´a de Medio Ambien e y O denacio´n del Te i o io, Comunidad de Mad id, license numbe 10/289334.9/07; Russia: Depa men o Rosp i odnadzo in S e dlo sk oblas and Council o Z enigo od Biological S a ion o Moscow S a e Uni e si y and bio-e hic codex o Biological Facul y o MSU, license numbe s N 212/647-249, Figu e 1. Loca ions o he popula ions sampled. G ey a ea illus a es b eeding ange o pied lyca che s in Eu ope (map modi ied om: Bi ds o he Wes e n Palea c ic, e sion 2.0.1., Ox o d Uni e si y p ess, 2003). FIN 1 = Ke o, Finland, NOR = Skibo n, No way; FIN 2 = Oulu, Finland; FIN 3 = Kauha a, Finland; FIN 4 = Ha ja al a, Finland; FIN 5 = Tu ku, Finland; EST, Pa ¨ nu, Es onia; SWE = O ¨land, Sweden; RUS 1 = Re da, Russia; LV = K asla a, La ia; RUS 2 = Moscow, Russia; GER 1 = Lingen, Ge many; UK = Powys, Uni ed Kingdom; NL = Buunde kamp, The Ne he lands; GER 2 = Ha z, Ge many; ESP = Lozoya, Spain. doi:10.1371/jou nal.pone.0025360.g001 Geog aphical Va ia ion in Egg Con en PLoS ONE | www.plosone.o g 3 No embe 2011 | Volume 6 | Issue 11 | e25360 p o ocol #04 No embe 16, 2005; NL: Animal Expe imen al Commi ee o he KNAW, license numbe DEC p o ocol no CTE 07-04; La ia: E hical Commi ee o Resea ch in Ecology and E olu ion, Dauga pils Uni e si y, license numbe 4/ZOO-2009; Ge many: Landk eis Gosla , Niede sachsen, Bundes epublik Deu schland, license numbe Az. 66 24 03-2.3.1.1; Finland and no he n No way (Lapland): Wes Finland Regional En i onmen Cen e, En i onmen al cen e o Sou hwes Finland, En i on- men al cen e o No h Os obo hia and En i onmen al cen e o Lapland, license numbe s LSU-2006-L-509 (254), LOS-2007-L- 264-254, PPO-2007-L-400-254 and LAP-2007-L-261-254; Es o- nia: Es onian En i onmen al Boa d, license numbe 1-4.1/11/ 100). Labo a o y p o ocol Yolk and albumen we e ca e ully sepa a ed in he labo a o y. Yolks we e weighed (,0.1 mg) and mechanically homogenised (as he dis ibu ion o e.g. ho mones may a y among egg laye s) [69,70]. App oxima ely hal o he yolk was used o and ogen analysis and J o bo h immunoglobulin and ca o enoid analyses. Yolks we e ozen a 220uC. All samples we e analysed simul aneously and by he same labo a o y, o a oid p oblems due o di e en analysis me hods. Immunoglobulin and lysozyme analyses we e conduc ed a he Depa men o Biological and En i onmen al Science, Uni e si y o Jy a¨skyla¨, Finland. Ca o - enoid analyses we e conduc ed a he Depa men o Chemis y, Uni e si y o Tu ku, Finland. Ho mone analyses we e conduc ed a he Uni e si y o Ve e ina y Medicine, Vienna, Aus ia. Immunoglobulin (IgG) analysis An ibody concen a ions we e de e mined using an indi ec enzyme-linked immuno-so ben assay (ELISA). The me hod is as desc ibed in [71]. B ie ly, he yolk samples we e weighed and dilu ed in 1:3 o dis illed wa e . The samples we e cen i uged a 13000 g o 15 min (a +4uC), in o de o sepa a e he clea immunoglobulin supe na an om he p ecipi a ed ex a ma e ial. The supe na an s we e collec ed and dilu ed (1:2000) in 1% BSA- PBS. The s anda d s ock solu ion was made by pooling an equal olume (5 ml) o he supe na an o each yolk sample (N = 351) and gi ing he undilu ed cock ail an a bi a y concen a ion o one million uni s o immunoglobulin pe ml (1000000 U/ml). A e he s anda d was ozen wi h glyce ol (1:1), he inal concen a ion o he s anda d s ock was 125 000 U/ml. The wells we e coa ed wi h 50 ml an i-chicken IgG (Sigma C- 6409, whole molecule, p oduced in abbi ) in ca bona e bu e o e nigh (o/n) a +4uC. He ea e he wells we e masked wi h 100 ml 1% BSA-PBS (1 h a oom empe a u e, RT). Duplica es o samples and s anda ds (50 ml) we e incuba ed h ee hou s in RT. The conjuga ed seconda y an ibody (Sigma A-9171, an i-chicken IgG whole molecule, alkaline phospha ase conjuga ed, 1:2000 dilu ion) (50 ml) was added and incuba ed (o/n) a +4uC. A e adding he subs a e (100 ml) (p-ni o phenyl phospha e, Sigma 104-0 in 1 M die hanolamine bu e ) he abso bances we e measu ed a 405 nm (Mul iskan Ascen , The ma Oy, Finland). The wells we e washed h ee imes wi h 0.05% Tween 20 in 16 PBS be ween he s eps ( i s wo washes 200 ml, hi d wash 400 ml). In e -assay a ia ion was 8.53% and in a-assay a ia ion 7.56%. Lysozyme analysis A mic o-pla e modi ica ion o he u bidime ic assay [72] was used o de e mine lysozyme ac i i y as desc ibed in [73]. Sho ly, albumen was dilu ed in phospha e bu e (67 mM, pH 6.2, dilu ion 1:500). A Mic ococcus lysodeik icus (Sigma M-3770) suspen- sion was p epa ed in phospha e bu e (0.5 mg/ml). The lysozyme o he samples will s a deg ading he bac e ial cell walls, which can be seen as clea ing o he Mic ococcus suspension and measu ed as change in abso bance wi h a mic opla e eade (Mul iskan Ascen , The ma Oy, Finland). 100 ml o dilu ed albumen and 100 mlo Mic ococcus we e added o he wells on he pla e and he abso bance was measu ed a 450 nm in oom empe a u e o 30 min using duplica es o samples. Be o e each measu emen , he pla e was mixed o 10 s. The esul s a e gi en as lysozyme ac i i y = change in abso bance uni s 61000/min (he ea e Dabs 61000/min). The linea pa o he declining cu e was used o calcula e he change in abso bance. In e -assay a ia ion was 5.6% and in a-assay a ia ion was 2.0%. And ogen analysis Fo measu ing he concen a ions yolk es os e one (T) and and os enedione (A4), we used a me hod simila o ha desc ibed in [69,74]. To ex ac s e oids, a e hawing, each yolk sample was suspended in 400 ml o dis illed wa e and 1600 ml me hanol and o exed wice o 30 s. Samples we e hen s o ed o e nigh a 4uC. Samples we e hen o exed and 1 ml o he suspension was ans e ed in o a new ial. The suspension was hen dilu ed wi h 1:5 assaybu e , o exed o 30 min and s o ed a 220uC o e nigh o p ecipi a e apola lipids. A e cen i uga ion (215uC, 2500 g, 10 min) 20 ml o he supe na an we e used o enzyme immunoassays. Fo ull desc ip ions o an ibodies and alida ion see [70,74–76]. In e -assay a ia ion was 9.9% (low le el pool) and 5.5% (high le el pool) o es os e one and 12.9% and 9.3% o and os enedione. In a-assay a ia ion was 7.9% o es os e one and 10.1% o and os enedione. Ca o enoid analysis Yolk ca o enoid concen a ions we e measu ed using a me hod simila o ha desc ibed in [77]. Fo ca o enoid analyses ca. 10 eggs om each popula ion we e andomly chosen (due o ime and inancial cons ains). Egg yolk was eeze-d ied (a 233uC o 48 h) and g ound in o ine powde . A known amoun o ine powde (app ox. 20 mg), was ex ac ed h ee imes wi h 100% ace one. The sol en was e apo a ed om he combined ex ac unde acuum and he esidue dissol ed in o a small olume o 100% ace one. The ca o enoid composi ion o he ex ac s was analysed wi h high-pe o mance liquid ch oma og aphy a 450 nm using an YMC C-30 (25064 mm, i.d., 5 mm) column and a g adien om 86% aqueous ace one in o 97% aqueous ace one ( low a e 1.5 ml/min). b-ca o ene was quan i ied using comme cial b-ca o ene as a s anda d and he o he ca o enoids (lu ein, zeaxan hin, o he xan hophylls and uniden i ied ca o en- oids) using comme cial lu ein as a s anda d. All he s anda ds we e pu chased om Ex asyn hese (F ance). Sum o all ca o enoids was used in he analyses ( o al ca o enoid concen a ion, mg/g). Ca o enoid p o iles ha e been desc ibed in [78]. Popula ion backg ound da a In addi ion o da a om he indi idual nes s om which eggs we e collec ed, backg ound da a om he s udy popula ions was collec ed (Table S1). This da a included coo dina es o he popula ions (la i ude and longi ude) and habi a ype da a (coni e ous o es , N = 7 popula ions; deciduous o es , N = 6 popula ions; mixed o es , N = 4 popula ions). S a is ical analyses All s a is ical analyses we e conduc ed wi h SAS 9.2. Lysozyme enzyme ac i i y was squa ed and concen a ions o yolk o al immunoglobulin, yolk es os e one, yolk and os enedione and yolk Geog aphical Va ia ion in Egg Con en PLoS ONE | www.plosone.o g 4 No embe 2011 | Volume 6 | Issue 11 | e25360 Geog aphical Va ia ion in Egg Con en PLoS ONE | www.plosone.o g 5 No embe 2011 | Volume 6 | Issue 11 | e25360 ca o enoids we e log- ans o med o no mali y. Fi s we quan i- ied among- and wi hin-popula ion a ia ion in egg componen s using simple Gene al Linea Models (GLM) in which he egg componen was he esponse a iable and popula ion he explana o y a iable. We hen s udied geog aphic a ia ion in egg componen s wi h linea mixed models (MIXED). The independen ac o s in he models we e: la i ude, longi ude, 2 nd o de e ms o la i ude and longi ude, habi a (coni e ous, mixed and deciduous), habi a 6la i ude and habi a 6longi ude. We in es iga ed he e ec o longi ude (along wi h la i ude) on egg componen s as he clima ic con inen ali y g adien ac oss Eu ope (a ec ing ain all and empe a u es) may also a ec en i onmen al a iables impo an in de e mining alloca ion o egg componen s. The analyses o speci ic ca o enoid p o iles and o al ca o enoids a e epo ed elsewhe e [78], bu he analyses o o al ca o enoids a e p esen ed also he e o a compa ison wi h o he egg componen s. Popula ion was included as a andom ac o o accoun o po en ial non-independence o he samples om one popula ion. Since some o ou sampling si es we e close o one ano he han o he s, we i s checked whe he he e was spa ial au oco ela ion in model esiduals. Mo an’s I coe icien s anged om 20.019 o 0.022 (N = 155 o ca o enoids and 333–349 o o he egg componen s), indica ing no spa ial au oco ela ion, hus a de aul co a iance s uc u e ( a iance componen s) was used. Non-signi ican e ms we e d opped om he models one-by-one, s a ing om he in e ac ions. The d opped non-signi ican main e ec s and in e ac ions we e again added in he educed models one a a ime and s a is ics a e e-in oducing hem in o he inal model a e epo ed. We calcula ed Spea man co ela ions among he a e age alues o egg componen s and a e age b eeding pa ame e s (laying da e, clu ch size, ha ching success, and ledging success) o each popula ion (nes s whe e eggs we e collec ed). We u he analyzed co a ia ion among he egg componen s using linea mixed models (p oc MIXED) by assigning each egg componen as esponse and explana o y a iable a a ime, and including popula ion as a andom ac o , accoun ing o non- independence o he samples om each popula ion. Resul s Among-popula ion s. wi hin-popula ion a ia ion in egg componen s Popula ion a e ages and ange o a ia ion in he egg componen s in each popula ion a e shown in Figu e 2A–G and Table S1. The e was signi ican a ia ion among popula ions in each egg componen wi h he excep ion o egg mass (all o he p- alues#0.01; Table 1). In yolk mass, albumen lysozyme ac i i y, yolk immunoglobulin concen a ion, yolk es os e one and yolk and os enedione concen a ions popula ion explained a ound 10% o he o al a ia ion. In yolk ca o enoid concen a ion, popula ion explained a ound 40% o he o al a ia ion (Table 1). Fo compa ison, popula ion explained 84% o a ia ion laying da e and 12% in clu ch size, espec i ely. Geog aphic a ia ion in egg componen s The e we e no geog aphic ends (ei he linea o quad a ic) in any o he egg componen s, wi h he excep ion o ca o enoids (Table 2). To al ca o enoid concen a ion showed a quad a ic end wi h la i ude (b6SE = 20.0007960.0002, Table 2, Fig. 2G): Ca o enoid concen a ion appea ed o be low in he mos sou he ly popula ion (Spain), highes in Cen al Eu ope (i.e. 50–55uN) and dec eased again owa ds he no h. To al ca o enoid concen a ion showed also a linea dec ease end om wes o eas (b6SE = 20.00360.0013, Table 2, see also e 78 o pa e ns in ca o enoid composi ion and p o iles). None o he egg componen s di e ed among habi a s (Table 2). Co ela ions among egg componen s and b eeding pa ame e s a he popula ion le el A e ages o b eeding pa ame e s in he nes s whe e eggs we e collec ed a e p esen ed in Table S1. We ound ha laying da e was weakly nega i ely co ela ed wi h ca o enoid concen a ion ( s =20.43, p = 0.08, N = 17) bu no wi h any o he o he egg componen s (20.32, s ,0.13, p.0.30, N = 17). Clu ch size was posi i ely co ela ed wi h es os e one le els ( s = 0.5, p = 0.03, N = 16) bu no wi h any o he egg componen (20.12, s ,0.12, p.0.33, N = 16). None o he egg componen s was co ela ed wi h ha ching success (20.40, s ,0.15, p.0.08, N = 14). Lysozyme ac i i y and immunoglobulin concen a ion we e nega i ely co ela ed wi h ledging success (lysozyme: s =20.63, p = 0.02 and IgG: s =20.79, p = 0.0013, espec i ely, N = 13 in bo h). Fledging success was no co ela ed wi h any o he egg componen (20.37, s ,0.37, p.0.20, N = 13). Co ela ions among egg componen s Egg mass and yolk mass we e posi i ely co ela ed ac oss he popula ions (Table 3), as well as es os e one and and os enedione concen a ions (Table 3). Lysozyme ac i i y was weakly nega i ely co ela ed wi h egg mass (Table 3). The e we e no o he signi ican co ela ions be ween he measu ed egg componen s (Table 3). Discussion We ound ha he e was signi ican a ia ion among pied lyca che popula ions in all he measu ed egg componen s, wi h he excep ion o egg mass. Howe e , popula ion explained only a Figu e 2. A–G. Among-popula ion a ia ion in he measu ed egg componen s. Among-popula ion a ia ion (mean 6SE) in ela ion o la i ude in he measu ed egg componen s. Abb e ia ions o each coun y and loca ion a e he ollowing: FIN 1 = Ke o, Finland, NOR = Skibo n, No way; FIN 2 = Oulu, Finland; FIN 3 = Kauha a, Finland; FIN 4 = Ha ja al a, Finland; FIN 5 = Tu ku, Finland; EST, Pa ¨ nu, Es onia; SWE = O ¨land, Sweden; RUS 1 = Re da, Russia; LV = K asla a, La ia; RUS 2 = Moscow, Russia; GER 1 = Lingen, Ge many; UK = Powys, Uni ed Kingdom; NL = Buunde kamp, The Ne he lands; GER 2 = Ha z, Ge many; ESP = Lozoya, Spain. Sample sizes a e shown in Table S1. doi:10.1371/jou nal.pone.0025360.g002 Table 1. Among-popula ion (N = 16 popula ions) a ia ion in egg size and egg componen s. Egg componen R 2 Fp N Egg mass (g) 0.03 0.75 0.73 342 Yolk mass (mg) 0.11 2.84 ,0.001 349 Albumen lysozyme ac i i y (Dabs 61000/min) 0.22 6.10 ,.0001 347 Yolk immunoglobulins (U/ml) 0.11 2.63 0.01 351 Yolk es os e one (ng/mg) 0.10 2.41 ,0.01 351 Yolk and os enedione (ng/mg) 0.12 3.24 ,.0001 351 Yolk o al ca o enoids (mg/g) 0.39 6.28 ,.0001 162 Resul s a e om a GLM wi h popula ion as he explana o y a iable. doi:10.1371/jou nal.pone.0025360. 001 Geog aphical Va ia ion in Egg Con en PLoS ONE | www.plosone.o g 6 No embe 2011 | Volume 6 | Issue 11 | e25360 small pa o he o al a ia ion in he egg componen s (wi h he excep ion o ca o enoids). Thus he majo i y o a ia ion was ound among indi iduals wi hin popula ions. We ound no geog aphical ends in any egg componen s, wi h he excep ion o ca o enoids. The low among-popula ion a ia ion ound o he egg componen s may indica e ha he bene i s o di e en ial alloca ion do no di e among en i onmen s and ha he e is no s ong local selec ion on he alloca ion o esou ces o eggs, despi e la ge en i onmen al di e ences. A ecen gene ic analysis also e ealed ha pied lyca che popula ions, especially in he No he n and Eas e n Eu ope, a e no di e en ia ed om each o he , sugges ing ex ensi e gene low and li le scope o local adap a ions [79]. Thus he po en ial o egg componen s o play a ole in popula ion di e gence in o sp ing ai s may no be s ong, al hough i has been shown o some o he ma e nal e ec s [37,42,44,80]. Howe e , he e a e also al e na i e pa hways which could acili a e he ole o ma e nal e ec s ia eggs in ai e olu ion and popula ion di e gence. Thus popula ions may di e in wi hin- clu ch pa e ns o deposi ion [81] o in ela ionships be ween egg componen s. An excep ion o he gene al pa e n o low among-popula ion a ia ion was ca o enoids, in which he among-popula ion a ia ion was conside ably highe (40%) han in o he egg componen s. Ca o enoid concen a ions appea o ha e only a mino gene ic componen [46,82], and hei le els in he yolk a e mainly de e mined by ca o enoid a ailabili y in he mo he ’s die [58,60,83]. The la ge among-popula ion a ia ion is he e o e likely mainly due o a ia ion in ca o enoid a ailabili y in di e en en i onmen s o di e ences in hei abso p ion o ans e o yolk. Thus a ia ion in ca o enoid le els mos likely e lec s pheno ypic ( esou ce-dependen ) a he han gene ic a ia ion. Yolk ca o en- oid le els ha e been ound o a y among popula ions also in o he species [36,58,60]. Wha explains he ex ensi e wi hin-popula ion a ia ion in esou ce deposi ion o eggs? P e ious s udies indica e ha deposi ion o se e al egg componen s is associa ed wi h en i on- men al o social condi ions wi hin popula ions: Fo example, Table 3. Co a ia ion among egg componen s. Yolk mass Lysozyme IgG T A4 Ca o enoids Egg mass 1.8260.14 20.3560.17 28.56623.51 12.46615.05 16.54622.32 25.31686.36 F 1, 336 = 162.12*** F 1, 339 = 4.37* F 1 ,254 = 1.30 F 1, 310 = 0.69 F 1, 276 =0.55 F 1, 154 = 0.09 Yolk mass 20.1160.056 5.5167.41 6.5164.66 5.8067.00 211.24625.87 F 1,330 = 3.7 F 1, 347 = 0.55 F 1, 346 = 1.96 F 1, 346 =0.69 F 1, 116 = 0.19 Lysozyme 6.1267.03 7.4664.47 11.4966.70 225.86627.7 F 1, 340 = 0.76 F 1, 341 = 2.80 F 1, 342 =2.90 F 1,150 = 0.87 IgG 20.0160.04 20.0160.05 0.0160.22 F 1, 349 = 0.19 F 1,348 =0.07 F 1, 123 = 0.00 T0.6160.07 0.3860.36 F 1, 349 = 70.41*** F 1, 101 = 1.10 A4 20.0460.23 F 1, 142 = 0.03 ***,0.001, **,0.01, *,0.05. Reg ession coe icien s (6SE) and F- alues among he measu ed egg componen s ac oss whole da a. Popula ion was included as a andom ac o o con ol o non- independence o he samples om each popula ion. IgG = yolk immunoglobulins, T = yolk es os e one, A4 = yolk and os enedione. doi:10.1371/jou nal.pone.0025360. 003 Table 2. Geog aphical a ia ion in egg size and egg componen s. Egg mass Yolk mass Squa ed lysozyme Log IgG Log T Log A4 Log ca o enoids Expl. a iables DDF F DDF F DDF F DDF F DDF F DDF F DDF F La i ude 17.1 2.1 13.8 1.32 14.2 0.04 15 0.25 12.5 0.08 12.7 0.02 12.9 10.59 a Longi ude 17.3 1.08 12.7 0.15 13.4 2.83 14.3 0.45 11.1 3.68 12 1.03 12 6.73 b Quad a ic la i ude 14 1.18 13.5 0.64 13.3 0.03 13.9 0.08 11 0.55 11.8 0.1 13.1 11.2 c Quad a ic longi ude 11.2 1.24 11.5 0.25 12.0 0.75 12.8 0.67 9.74 0.19 10.9 0.03 10.7 0.51 Habi a 22.5 1.22 19 0.09 24.9 0.16 21.7 0.32 16.4 0.99 18.2 0.51 16.8 2.28 La i ude 6habi a 25.4 1.39 20.9 0.11 25.2 0.25 13.1 2.21 17.3 0.55 18.8 0.53 10.5 1.97 Longi ude 6habi a 4.01 1.53 20.8 0.01 25.8 0.36 13 0.71 17.8 0.22 20.3 0.14 8.92 0.14 Resul s a e om linea mixed models explaining geog aphical a ia ion in egg componen s. Popula ion was included as a andom e ec in all models. The only signi ican e ec s a e indica ed wi h bold and le e s (a–c). IgG = yolk immunoglobulins, A4 = yolk and os enedione, T = yolk es os e one.Nume a o d is 2 o habi a and 1 o o he explana o y a iables. a: p = 0.006, b: p = 0.023, c: p = 0.005. doi:10.1371/jou nal.pone.0025360. 002 Geog aphical Va ia ion in Egg Con en PLoS ONE | www.plosone.o g 7 No embe 2011 | Volume 6 | Issue 11 | e25360 deposi ion o and ogens in he s udy species as well as in o he species has been associa ed wi h iming o b eeding [61,62], ood a ailabili y (Laaksonen, T. unpublished), b eeding densi y o emale o male quali y [25,47,59,61–63]. Deposi ion o an ibodies and an ibac e ial enzymes has been associa ed wi h iming o b eeding [59], pa asi e load, emale condi ion and ma e quali y [34,57,59,84]. Thus i may be ha a ia ion in he ele an en i onmen al ac o s (especially emale and ma e quali y) a ec ing deposi ion o egg componen s is simply la ge wi hin han among popula ions. Fu he mo e, di e ences in ep oduc i e physiology o gene ic ac o s among indi iduals wi hin popula ions may play a ole [45,58–63]. Egg size and deposi ion o se e al egg componen s (yolk mass, yolk immunoglobulins, yolk es os e one, po en ially lysozyme) indeed ha e been ound o ha e a gene ic componen [27,45–47]. Howe e , as his is he i s ime in a- speci ic a ia ion in ma e nal e ec s in eggs has been s udied on a la ge scale, mo e s udies quan i ying among- and wi hin- popula ion a ia ion a e needed o e eal pa e ns in o he species and he e olu iona y po en ial o ma e nal e ec s in eggs. In con as o ou hypo hesis, egg quali y, measu ed in he o m o di e en egg componen s, did no gene ally a y geog aphically, despi e he la ge la i udinal changes in o example empe a u e du ing egg-laying, du a ion o he b eeding season, p edic abili y o en i onmen al condi ions and po en ially also in ood supply. This may imply ha deposi ion o di e en egg componen s is simply no e lec ing inc easing egg quali y (sensu li e-his o y heo y), and bene i s o di e en ial alloca ion may no di e among popula ions. Al e na i ely, e en i highe esou ce deposi ion would be bene icial o he o sp ing, emales may be cons ained in hei deposi ion o eggs, due o cos s o hemsel es [18,25,45]. We may also specula e ha pe haps he ele an en i onmen al and social ac o s (e.g. emale and ma e quali y, iming o b eeding, b eeding densi ies and pa asi e exposu e, see abo e) ha a e ound o a ec deposi ion o egg componen s in he s udy species [59–63], do no show geog aphical ends, bu a y mo e wi hin han among popula ions. We u he ound ha , a he popula ion le el, iming o b eeding, clu ch size o ha ching success did no seem o be associa ed wi h deposi ion o he egg componen s. Only ca o enoids ( o al ca o enoids and he p opo - ion o lu ein and o he xan ophylls), showed geog aphical ends (ei he linea o quad a ic), dec easing om Cen al Eu ope owa ds he no h [78]. This mos likely e lec s he a ailabili y o ca o enoid- ich ood (especially lepidop e an la ae), which is lowe in no h due o no he n popula ions s a ing egg-laying ea lie ela i e o he ee phenology han cen al Eu opean popula ions [78]. None o he egg componen s, wi h he excep ion o speci ic ca o enoids (lu ein and o he xan hophylls) [78] a ied among di e en habi a s, al hough a ia ion in o example ood quali y and quan i y among habi a s has been shown [85,86]. In con as o ou hypo hesis, we ound gene ally no co ela ions among egg componen s ac oss he whole da a se . Only egg mass and yolk mass as well as he wo and ogen ho mones we e s ongly co ela ed, as expec ed on he basis o p e ious s udies [45,47]. Albumen lysozyme enzyme ac i i y was nega i ely co ela ed wi h egg mass, a esul suppo ed by some p e ious s udies [87], sugges ing a po en ial cos o cons ain in alloca ing lysozyme o eggs. Ou esul s a e also consis en wi h ea lie s udies [64,88] showing ha indi idual egg le el o clu ch le el co ela ions be ween egg mass, an ioxidan s, immunoglobulins and and ogens we e no ound despi e o co ela ed wi hin-clu ch a ia ion. The lack o co ela ion among hese compounds may simply e lec ha hey a e egula ed by di e en p ocesses. Howe e , co- a ia ion among egg componen s may di e in di e en en i onmen s, o example due o di e en ial ade-o s in alloca ion be ween sel and o sp ing in ela ion o ood a ailabili y and condi ion o p e alence o in ec ious diseases. This issue should be u he in es iga ed. Conclusions We ound ha he e was signi ican a ia ion among popula- ions in mos egg componen s, bu mos o he a ia ion was among indi iduals wi hin popula ions, p obably due o high plas ici y in deposi ion o eggs. No geog aphic pa e ns in egg quali y, wi h he excep ion o ca o enoids, we e ound. As ou s udy was explo a o y, mo e s udies a e needed o ho oughly unde s and he ole o ma e nal e ec s in e olu iona y change. Suppo ing In o ma ion Table S1 Among-popula ion a ia ion in b eeding pa ame e s and egg componen s in he pied lyca che . Mean (6SD), minimum and maximum alues o he measu ed egg componen s o each s udy popula ion a e shown along wi h he loca ions o he s udy popula ions, habi a ypes and a e ages o b eeding pa ame e s o he nes s whe e eggs we e collec ed. La = la i ude (uN), Long = longi ude (uE), N = sample size pe popula ion. Fo laying da e 1 = 1.4.2007. (DOC) Acknowledgmen s We hank lab echnicians a he Uni e si y o Jy a¨skyla¨ and all he people p o iding help in ield wo k in di e en popula ions. We also hank he anonymous e e ees o hei aluable commen s. Au ho Con ibu ions Concei ed and designed he expe imen s: SR TL. Pe o med he expe imen s: SR TE EB AJ AK IK JM CM RM MO AQ FS VT MEV WW HZ TL. Analyzed he da a: SR TL TE. W o e he pape : SR HS TE EB AJ AK IK JM CM RM EM MO AQ JPS FS VT MEV WW HZ TL. Labo a o y analyses: SR EM HS JPS. Collec ed he da a: SR TE EB AJ AK IK JM CM RM MO AQ FS VT MEV WW HZ TL. Re e ences 1. May E (1956) Geog aphical cha ac e g adien s and clima ic adap a ion. E olu ion 10: 105–108. 2. Endle JA (1977) Geog aphic a ia ion, specia ion, and clines. P ince on Uni P ess. 3. Sla kin M (1973) Gene low and selec ion in a cline. Gene ics 75: 733–756. 4. Li B, Suzuki JI, Ha a T (1998) La i udinal a ia ion in plan size and ela i e g ow h a e in A abidopsis haliana. Oecologia 115: 293–301. 5. Mo ison C, He o JM (2003) Geog aphic a ia ion in li e-his o y cha ac e is ics o amphibians: a e iew. J Anim Ecol 72: 270–279. 6. Kawecki TJ, Ebe D (2004) Concep ual issues in local adap a ion. Ecol Le 7: 1225–1241. 7. Aze edo RBR, F ench V, Pa idge L (1996) The mal e olu ion o egg size in D osophila melanogas e . E olu ion 50: 2338–2345. 8. Rowe JW (1994) Rep oduc i e a ia ion and he egg size-clu ch size ade-o wi hin and among popula ions o pain ed u les (Ch ysemys pic a bellii). Oecologia 99: 35–44. 9. Shine R (2003) Rep oduc i e s a egies in snakes. P oc R Soc Lond B 1519: 995–1004. 10. Johns on TA, Legge WC (2002) Ma e nal and en i onmen al g adien s in he egg size o an i e opa ous ish. Ecology 83: 1777–1791. 11. Lack D (1947) The signi icance o clu ch size. lbis 89: 302–352. 12. Klomp H (1970) The de e mina ion o clu ch size in bi ds. A e iew. A dea 58: 1–124. 13. God ay HCJ, Pa idge L, Ha ey PH (1991) Clu ch size. Ann Re Ecol Sys 22: 409–429. 14. Monaghan P, Nage R (1997) Why don’ bi ds lay mo e eggs? T ends Ecol E ol 12: 270–274. Geog aphical Va ia ion in Egg Con en PLoS ONE | www.plosone.o g 8 No embe 2011 | Volume 6 | Issue 11 | e25360