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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
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Geog aphical Va ia ion in Egg Con en
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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
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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
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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
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PLoS ONE | www.plosone.o g 8 No embe 2011 | Volume 6 | Issue 11 | e25360