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RESEARCHARTICLE
Concertedevolutionofbodymassandcellsize:similarpatternsamong speciesofbirds(Galliformes)andmammals(Rodentia)
MarcinCzarnoleski1,*,AnnaMariaLabecka1,DominikaDragosz-
Kluska1,TomaszPis1,KatarzynaPawlik1,FilipKapustka1,WincentyM.Kilarski2a n dJanKozłowski1
ABSTRACT
Cells i z e p l aysa rolei n b o d y s i z e e volutiona n d e n v i ronment aladaptations.Addressingtheseroles,westudiedbodymassandcellsizei n G a l l i f o r m e s b i r d s a n d Rodentiam a m m a l s , a n d c o l l e c t e d publisheddataontheirgenomesizes.Inbirds,wemeasurederythr ocytenucleiandbasalmetabolicrates(BMRs).Inbirdsandmam mals,largerspeciesconsistentlyevolvedlargercellsforfivecelltypes(erythrocy tes,enterocytes,chondrocytes,skinepithelialcells,andkidneyproximalt ubulecells)andevolvedsmallerhepatocytes.Wefoundnoevidenceth atcellsizedifferencesoriginatedthroughgenomes i z e c h a n g e s . Wec o n c l u d e t h att h e o r g a n i s m -
w i d e coordinationo f c e l l s i z e c h a n g e s m i g h t b e a n e volutiona rilyconservativecharacteristic,a n d theco nvergente volutionaryb o d y sizeandcellsizechangesinGalliformesandRodentiasuggesttheada ptivesignificanceofcellsize.Recenttheorypredictsthatspeciesevolvinglarge rcellswastelessenergyontissuemaintenancebuthavereducedc a p acitiest o d e l i vero x y g e n t o m i t o c h o n d r i a a n d metab olizeresources.I n d e e d , b i r d s w i t h l a r g e r s i z e o f t h e abov ementionedcelltypesandsmallerhep atocyteshaveevolvedlower mass-
specificBMRs.Weproposethattheinconsistentpatterninh e p atocytesd e r i vesf romt h e e f f i c i e n t d e l i verysystemt o hepatocytes,c o m b i n e d w i t h t h e i r i n t e n s e i n volvementi n supracellularfunctio nandanabolicactivity.
KEYWORDS:Allometry,BMR,Bodysize,Concertedevolution,Interspec ificscaling,Karyoplasmicratio,Metabolicrate,Optimalcellsize,Species diversity
INTRODUCTION
Whethertheyarebacteria,protists,fungi,plantsoranimals,livingthing sh avee volveda p l e thorao f d i f f e rentb o d y p l a n s a n d l i f e str ategies,resultingindramaticdifferencesin bodymassamong spe cies.Weknowsurprisinglylittleaboutthecellularmechanismsinvolvedi ntheoriginofthisvariance.Theevolutionoflargerorsmallerorgan ismscanoccursimplythroughchangesincellnumber,whichshould helppreservethefundamentalphysiologicalcharacteristicsofsinglecel lsinabody.However,achangeincellnumbermayaffectphysiological performanceifthenumberofcells
1InstituteofEnvironmentalSciences,JagiellonianUniversity,Gronostajowa7,30- 387Kraków,Poland.2InstituteofZoology,DepartmentofBiologyandCellImaging,Jagielloni anUniversity,Gronostajowa9,30-387Kraków,Poland.
*Authorforcorrespondence(marcin.czarnoleski@uj.edu.pl)M.
C.,0000-0003-2645-0360;A.M.L.,0000-0002-8810-7093
ThisisanOpenAccessarticledistributedunderthetermsoftheCreativeCommonsAttributionLicense(h ttp://creativecommons.org/licenses/by/3.0),whichpermitsunrestricteduse,distributionandreprod uctioninanymediumprovidedthattheoriginalworkisproperlyattributed.
Received6 September2 0 17 ; A c c e p te d 7 March2018
inanorganaffectsorganfunctionoriftissuemaintenancedependsoncellnu mberandcellsize.Accordingtothetheoryofoptimalcellsize(TOCS) (Atkinsonetal.,2006;Czarnoleskietal.,2015a,2016;Davison,1956;Kozłows kietal.,2003;Szarski,1983),cellsizeisoptimizedaccordingto th eor gan is m’srequirements,a nd i ts adaptivevaluedependsontheco stassociatedwiththemaintenanceofthecellmembraneandtheca pacityofthecelltoperformphysiologicalfunctions.Tomaintainthefuncti oningofcellmembranes,anorganismdevotessubstantialamountsofener gytothemaintenanceofthephysicalpropertiesofcellmembranes(England Attwell,2015)andtothegenerationofelectro-
chemicalpotentialsacrosstheirsurface(RolfeandBrown,1997).
Allelsebeingequal,energeticdemandperunitmassshould be lowerinlargerorganismsifbodymassevolvesinconcertwithcellsize.
Alargerbodythatconsistsofnotonlymorebutalsolargercellshasasmaller amountofcellmembranesperunitoftissuemass,whichshouldl ow eri t s metabolicc o stsp e r unito f b o d y m a s s . Nevertheless,or ganswithlargecellsareexpectedto metabolizeata s lowerratet h a n a reo r g a n s w i t h s m a l l c e l l s b e c a u s e o f t h e smallersurfacear eaofcellsavailablefortheexchangeofsubstratesandp roducts,t h e l o n g e r d i stancesi n volvedi n i n t racellulardiffusion,a n d t h e f ewer n u cleif o r t ranscriptioni n o r g answ i t h largecells(Czarnoleskieta l.,2015b).
Todate,theTOCShasbeenusedtoaddresstheoriginofcellsizevariancei n e c t o t hermsexposedt o e n v i ronmentalg radients(Czarnoleski etal.,2015b;Walczyńskaetal.,2015),andinectothermsa n d e n do thermsc h a racterizedby d i f f e rentm e tabolicrates(Hermaniuketal.,2 017;Maciaketal.,2011,2014;Starostováetal.,2013).Mostofthestudiesonth esetopicshavefocusedononecelltypeandgeneralizedtheirresultstoor ganism-
widetrendsincellsize,butsoundconclusionsregardingthecellulararc hitectureofanorganismrequireinformationaboutthecellsizesindiffere ntbodypa rt sa ndont he cell s i z e s o r i g i n atingfromd iff erentge rmlayers.Toaddressthisproblem,westudiedthebodymassandsizeofe rythrocytes,chondrocytes,hepatocytes,enterocytes,epithelialskinc ellsandkidneycellsindifferentspeciesofGalliformesbirdsandRodentia mammals.Wealsom e a s u redn u c l e i int h e erythrocytesofbird s,andusedthedatabasebyGregory(2017)toextractpublisheddatao nthegenomesizesofthestudiedspecieswhereavailable.Wefirstai medtoexaminewhetherspeciesdivergedw i t h respectt o c e l l s i z e a n d w h e t h e r t h i s d i vergenceinvolvedcoordinatedchangesi ncellsizeindifferenttissuetypes(hypothesisI ) . FollowingG re g o r y ( 20 0 1 ) a n d K o z łowskie t a l .
( 2 0 0 3 ) , wep redictedt h ats u c h e volutioni n volvesa l t e rationsi n genomesize.Toexplorethisidea,weexaminedlinksbetweenthesize so f c e l l s,n u c l e i a n d g e n o mes.Wea l s o c o m p a redt h e karyopl asmic(nu cleus-to-
cells i z e ) ratiosof e r y t h rocytesa m o n g birdspecies.Followingthee videnceofCavalier-
Smith(2005),weexpectednovarianceinthisratio.Next,wetestedw hetherinterspecificdifferencesinbodymassevolvedinassociationw ith
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changesincellsize(hypothesisII)orexclusivelythroughchangesinthen umberofcells.NotethatifhypothesisIholds,wemightthenexpectthatbodym assevolvedwithaninvolvementoforganism-
widechangesincellsize.Finally,focusingonbirds,wemeasured
Table1.Cellsizeunderwentcoordinatedchangesindifferentcelltypes,andthepat ternofthesechangeswassimilarinRodentiaandGalliformes
Organ/ Rodentia Galliformes
basalmetabolicrates(BMRs)inthesameindividualsforwhichwe
collectedinformationaboutcellsize.Inthisway,weexamineda Celltype tissue PC1 PC2 PC1 PC2
predictionregardingtheTOCSstatingthattheevolutionoflarge- celledspeciesshouldbeassociatedwithlowermass-
specificcostsoft i s suem a intenance( h y p o t h esisI I I )
( K o z łowskie t a l . , 2 0 0 3 ) . Notet h ati f h y p o t h esisI I h o l d s , t h e n f o l l owingh y p o t h e s i s I I I , BMRsshouldincreasewithb odymassataratiooflessthan1:1
Erythrocytes Blood 0.77 0.07 0.61 0.68
Enterocytes Duodenum 0.89 0.28 0.59 −0.58
Proximaltubulecells Kidney 0.90 0.30 0.88 −0.21
Epithelialcells Skin 0.99 0.13 0.72 0.27
Chondrocytes Trachea 0.89 −0.27 0.89 0.35
Hepatocytes Liver −0.61 0.77 −0.54 0.74
(negativeallometricscalingwithmass),whichwouldcorrespondto lowermass-specificBMRsinlargerspecies.
%ofexplained variance
72 14 52 26
RESULT S
Accordingtoourprincipalcomponentanalysis(PCA)
(Table1),inbothg roupso f a n i mals,t h e s izeso f e r y t h rocytes,e n t e rocytes,chondrocytes,e p i thelials k i n c e l l s a n d k i d n eyc e l l s l o a d e d positivelyonthefirstprincipalcomponent(PC1), andthesizeofhepatocytesloadednegativelyonPC1.Thispatternin dicatesthatacrossspecies,cellsinfivetissueshadpositivelycorrelat edsizes,andt h e s izeso f t h e s e c e l l s werei n verselyrelatedt o t h e s i z e o f hepatocytes.T h e s e c o n d p r i n c i p alc o m p onent( P C 2 ) m a i n l y explainedt h e p o s i t i vee f f ectso f h e p atocytes( m a m mals)o r t h e positiveeffectsofhepatocytesanderythrocytesandnegativeeff ectsofduodenalenterocytes(birds).Dataontherawmeasuresofcell sizeareprovidedinTableS1.
Intotal,thetwoprincipalcomponents(PCs)explained86%ofthe interspecificvarianceincellsizeinmammalsand78%inbirds.Themajorpar tofthisvariance,explainedbyPC1(72%inmammalsand52%inbirds) ,wasrelatedtointerspecificdifferencesinbodymass(Fig.1),asindicatedby positivecorrelationsbetweenthePC1scoresandbodymassinmamma ls(r=0.84,P=0.04)andbirds(r=0.97,P=0.006).Inotherwords,largerspecie shaveevolvedsmallerhepatocytes,butlargererythrocytes,chond rocytes,enterocytes,k id n eyc el ls a n d s k i n c e l l s . I nf o r m ationo n t h e correlationb e t weenb o d y m a s s a n d rawc e l l s i z e i s p rovi dedi n
Thenatureoftheserelationshipsisshownbytheloadingvaluesfromtheprincipalco mponentanalysisofcellsize.Principalcomponents(PCs)witheigenvalues>1arerep ortedhere.ScoresforPC1andPC2wereusedtointegrateinformationonthecoordin atedchangesincellsizeandinstatisticalanalysestoexaminetheconcertedevolutionb etweencellsizeandadultmassamongspecies.
TableS2.ThepartofinterspecificvarianceincellsizeexplainedbyPC2(14%
inmammalsand26%inbirds)wasunrelatedtobodymass‒
PC2scoresdidnotcorrelatewithbodymass(mammals:r=0.14, P=0.79andbirds:r=0.02,P=0.98).
Inb i r d s , k a r yoplasmicratiosf o r e r y t h rocytesd i f f e redsign ificantlyamongspecies(F4,20=8.40,P=0.001).Themeansizeso f e r y t h rocytesa n d t h e i r n u cleiweren o t c o r relatedacrossspecies(r=
0.20,P=0.75;Fig.2A).Inthetwospeciesofbirdsforwhichweobtaine dinformationaboutgenomesize,thespecieswiththelargergenomehadslightl ylargererythrocytenucleiandsmallererythrocytes(Fig.2A),althought hisdifferenceismostlikelynotstatisticallysignificant.Inthefourspe ciesofmammalsforwhichweh a d i n f o r m ationo n g e n o mes i z e , g e n o m e s i z e wasn o t significantlyc o r relatedw i t h c e l l s i z e f o r P C 1 ( r = 0 . 5 7 , P =0.43;Fig.2B)orPC2(r=0.12,P=0.92).
Inbirds,BMRincreasedwithbodymass(r=0.996,P=0.0003;
Fig.3A).Themass-scalingexponentwas0.827,indicatinga
Fig.1 . I n b i r d s a n d m a m m a l s , l a r g e r s peciesh avee volveds m a l l e r h e p atocytesb u t l a r g e r e r y t h rocytes,c h o n d rocytes,e n t e r o c y t e s , k i d n eyc e l l s andskincells.Linesrepresentthestandardizedmajoraxis(mammals:y=−2.36+1.159x;birds:y=−4.72+1.940x).Symbolsrepresentthespeciesme anscalculatedfromthedataforfiveindividualsandaremarkedwithinitialsforeachspeciesname.Mammals:Ma,Microtusarvalis;Mc,Myocastorcoypus;Mg,
Myodesglareolus;Mm,Musmusculus;Ps,Phodopussungorus;Rn,Rattusnorvegicus.Birds:Ac,Alectorischukar;Cc,Coturnixchinensis;Cj,Coturnixjaponica;Pc,Phasi anuscolchicus;Pp,Perdixperdix.PC1isthefirstprincipalcomponentintheprincipalcomponentanalysisofcellsize.ScoresforPC1wereusedtointegrateinformationonthec
oordinatedchangesincellsize.Arrowsindicatetheloadingvaluesforcellsizeindifferentorgans/tissuesfromPC1(seeTable1),demonstratingthenatureofcellsizerelations hips.
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Fig.2 . I n b i r d s ( A ) , t h e s i z e o f e r y t h rocytesd i d n o t cor relatew i t h t h e s i z e o f e r y t h r o c y t e n u c l e i , a n d i n mammals( B ) , c e l l s i z e d i d n o t c o r r e l atew i t h g e n o m e size(C-value).Dataaboutgenomesize(C-
value;pg)wereavailableforonlytwobirdspecies,andtheyares howninAneartheinitialsofthespecies.C-
valueswerenotavailablefortwospeciesofmammals.Symbols arespeciesmeans,markedwiththeinitialsforeachspeciesnam e.PanelA:Ac,Alectorischukar;Cc,Coturnixchinensis;Cj,Cotu rnixjaponica;Pc,Phasianuscolchicus;Pp,Perdixperdix.PanelB :Mm,Musmusculus;Mc,Myocastorcoypus;Ma,Microtusarvali s;Rn,Rattusnorvegicus.PC1isthefirstprincipalcomponentinth eprincipalcomponentanalysisofcellsize.ScoresforPC1wereu sedtointegrateinformationonthecoordinatedchangesincellsiz e.Arrowsindicateloadingvaluesforcellsizeindifferentorgans/ti ssuesfromPC1(seeTable1),demonstratingthenatureofcellsiz erelationships.
negativeallometric relationship(0.703and0.973werethelower andu p p e r l i mitse stimateda s 9 5 % c o n f i d e n cei n t e r v a l s ) . M a s s -
specificm e t a bolicrateso f b i r d s weren e g ativelyrelatedt o P C 1 sc ores(r=−0.90,P=0.036;Fig.3B).Thus,largerbirdsthatevolvedsmallerh ep atocytesandlarg ererythrocytes,chond rocytes,enterocytes,s k i n c e l l s andkidneyc e l l s werec h a racterizedb y lowermass- specificmetabolicrates.Notethatalowresidual
varianceintherelationofBMRandPC1tobodymassmakesitdiffi cultt o exploret he m e tabolice ff e c t s o f P C 1 i n d e p e n dento f bod ymass.PC2scoreswerenotrelatedtomass-
specificmetabolicrates(r=0.16,P=0.80).
DISCUSSION
InGalliformesbirdsandRodentiamammals,specieswithalargerbody massareconsistentlycharacterizedbylargercellsforfivecelltypes(erythro cytes,enterocytes,chondrocytes,skinepithelialcells,kidneyp roximalt u b u l e c e l l s)a n d b y s m a l lerh e p atocytes.T h i s patterncalls atten tiontofourimportantph enomena,wh ich haveoftenbeenmisse dbyearlierstudiesbutcanhelpusgainabetterunderstandingofthen atureoftheevolutionaryprocessesthatdrivetheoriginofdifferencesintr aitsbetweenspecies.
Thefirstphenomenonwefounddemonstratesthatspecieshaveevo lvedc e l l s w i t h d i f f e rents i z e s , a n d t h i s e volutionaryc h a n g eshowsanorganism-
widedistribution,indicatingthatthecellular architectureofti ss u e sh a s evolvedin a coordinatedmann er throughouttheentirebod yratherthanoccurringonlyinindividualorgans.Previously,coordin atedchangesinthesizesofdifferenttypesofcellshaverarelybeenstudie d,buttheyhavebeenfound,eitheraspartofanevolutionaryorphenot ypicallyplasticprocess,inflies (A ze vedo etal., 2002;Czarnol eskiet al.,2016;Heinrichetal.,2011;Stevensonetal.,1995),reptiles(C zarnoleskietal.,2017),amphibians,birds,mammals(Kozłowskietal.,20 10;Maciaketal.,2014)andplants(Brodribbetal.,2013).Altogether,thise m e r g i n g e v i d e n c e s u g g e stst h ata n o r g a n i s m- widecoordinationo f c h a n gesi n c e l l s i z e m i g h t b e a n e volutio narilyconservedpropertyoforganisms.Atthemolecularlevel,theev olutionofcoordin atedchangesincellsizeisli kelytoinvolvealt erationsinthesignalingpathwaysthatcontrolandsynchronizecellula rgrowtha nd proliferationratesi n d iff erentt i s s u e s du r i ng develo pment,mostlikelytheTOR(targetofrapamycin)andinsulinregulatorypath ways(DeVirgilioandLoewith,2006;Grewal,2009;Montagneetal.,1999).Alt ernatively,butnotmutuallyexclusiveof
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Fig.3.Inbirds,metabolicrateincreasedallometricallywithbodymass
(A)andbirdswithsmallerhepatocytesandlargercellsintheotherfivetissu essampledhadlowermass-
specificmetabolicrates(B).Linesrepresentthestandardizedmajoraxis(A:y=1.
32+0.827x;B:y=7.11
−1.018x).Symbolsarespeciesmeanscalculatedfromdataforfive individualsandaremarkedwiththeinitialsforeachspeciesname:Ac,
Alectorischukar;Cc,Coturnixchinensis;Cj,Coturnixjaponica;Pc,Phasianuscolchic us;Pp,Perdixperdix.PC1isthefirstprincipalcomponentintheprincipalcomponentan alysisofcellsize.ScoresforPC1wereusedtointegrateinformationonthecoordinate dchangesincellsize.Arrows
indicateloadingvaluesforcellsizeindifferentorgans/tissuesfromPC1(see Table1),demonstratingthenatureofcellsizerelationships.
theroleofTOR/insulinsignaling,organism-
widechangesincellsizecanevolvethroughalterationsingenomes ize,e.g.viapolyploidizationorindel(insertion-
deletion)processes(Cavalier-
Smith,2005).Itishypothesizedthatcytologicalmechanismsregulat ethevolumeofacellbasedonthevolumeofitsnucleus.Giventhatth evolumeofanucleuslimitsthemaximumamountofDNAinacell,achange ingenomesizeshouldelicitachangeinthesizeofcellnuclei,whichshouldulti matelycorrespondtoachangeincellsize(ElliottandGregory,2015;G regory,2002;Kozłowskietal.,2003).Wearenotabletospecificallydeter
minewhichofthetwom e c h a n i s msp l ayeda m orei m p o r t a n t r olei n t h e c e l l u l a r
evolutionofthebirdsandmammalswestudied,buttheeffectsofgen omesizealoneseeminsufficienttoexplainthisevolutionarycha nge.Inbirds,wefoundevidencecontrarytotheideathatthesizeofc el ls evol vedintightassoci ationwi th th esizeofnucle i.Thekaryoplas micratiooferythrocytesdifferedbetweenspecies,andthesizeoferythroc ytesandtheirnucleiwerenotassociatedwitheachotheracrossspecies ,althoughweacknowledgethatt h e smallnumberofstudiedspecies mightdecreaseourstatisticalpowerfordetectings u cha n a s s o c i ation.N e vertheless,t h e s e i n t e r s p e cificpatternsdonotadhe retocommoncytologicalassumptionsaboutthetightassociationb etweenthesizesofcellsandtheirnucleiandthei n varianceo f k a r yopl asmicratiosi n n ature(Cavalier-
Smith,2005).Whenwecomparedspeciesofbirdsormammalsforwhic hweo b t a i n e d i n f o r m ationo n g e n o mes i z e , wed i d n o t f i n d a n y evidencethatlargercellsorcellnucleiwereassociatedwithl argergenomes.However,thesenegativeresultsshouldbetreated withcautionbecauseonlyasubsetofthestudiedspecieshadavailab ledataongenomesize.Additionally,thepublisheddataongenome sizewereo b t a i n e d f romd i f f e renti n d i v i d u a lst h a n t h e o n e s westudied,andcellsizeandgenomesizeareknowntovarynoto nlyamongb u t a l s o w i t h i n s p e c i e s . H owever,n o t f i n d i n g statisticalconnectionsbetweencellsizeandthesizeofgenomesorcellnucl eimightalsoberevealing–
itislikelythatsomespeciesevolvedcellsizedifferencesthroughalteratio nsingenomesize,whereasinotherspecies,theevolutionofcellsizeinvolved changesinthepropertiesoftheTOR/insulinpathwayswithoutachangei ngenomesize.Wespeculatethatifdecreasesandincreasesingenomesize donothaveequalconsequences(e.g.deletionsresultinahigherrisk ofgenelossthaninsertionsorgenomemultiplications;thenumberofge necopiesaffectsthebiochemicalfunctionofcells),thentheevolutionofc ellsizeinonedirectionwouldproceedmostlythroughchangesing e n o mes i z e , b u t c h a n gesi n t h e o t h e r d i rectionw o u l d o c c u r thro ughalterationsinTOR/insulinsignaling.
Oursecondfindingindicatesthatchangesincellsizeindifferentspecies evolvedintightconnectionwithchangesinbodymass;achangei ncellsizewasapartoftheme chanismin volvedintheevolution ofadultmass.Theroleofchangesincellsizeineitherevolutionary o rp h e n o t y p icallyp l a sticc h a n gesi n b o d y s i z e h a s beensug gestedbyearlierstudies(Adrianetal.,2016;Czarnoleskiet al., 2013, 2015b;H es sen et al., 2013 ;S ta rostováetal., 2005 ;Partrid geetal.,1999),butrarelyhasthisrolebeendemonstratedsimulta neouslywithreferencetoinformationaboutdifferentcelltypes(
b u t s e e K o z ł ow s k i e t a l . , 2 0 1 0 ; S t e vensone t a l . , 1 9 9 5 ) . Unlikeeutelicorganisms,whichhaveconstantcellnumbers,such asrotifers,n e m atodesa n d s p r i n g t a i ls( VanVoorhies,1 9 9 6 ; W a l c z y n ́ s k a e t a l . , 2 0 1 5 ) , d e velopmentalc o nstraintsl i n k i n g t h e growtho f a b o d y w i t h t h e g rowtho f c e l l s c a n n o t explaint h e concertedevolutionarychangesincellsizeandbodymas sinnon-
eutelicvertebrates.Theresultsofan artificialselect ion studyon micede mon stratedt h atce ll s i z e i n d i f f e rentti s suesh a s e volv edindependentlyofbodymass(Maciaketal.,2014),indicatingth atcellsizeandbodymassinnon-
eutelicorganismshavethefreedomtoevolveindependently.
Accordingtoourthirdfinding,cellsizeandbodysizeevolvedinconcert i n a s i m i l a r m a n n e r i n b i r d s a n d m a m m a l s d e s p i t e t h e independentevolutionaryhistoriesofbothg roups.Weviewth isevolutionaryconvergenceasanindicationthatconcertedevolutionb etweencellsizeandbodymassisnotneutral,demonstratingtheeffe ctsofnaturalselectionratherthanrandomchanges.Supportingthisadap tiveview,DrosophilamelanogasterMeigen,1830evolvedsimilarlatit udinalc l i n e s inc e l l size,bodys ize,andt h e characteristicso f t h e i r TOR/insulinp athwayso n t h e A u stralian
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andNorthAmericancontinentsdespitetheindependentoriginsofthet w o c l i n e s ( D e J o n g a n d B o c h d a n ov i t s , 2 0 0 3 ; Fabiane t a l . , 2012;Paabyetal.,2010).Questionsremainregardingthistopic.Forexample, whataretheselectiveadvantagesanddisadvantagesofagivencellsize, andwhyhavelargerspeciesevolvedlargercells?
Basedo n i n f o r m ationa b o u t t h e B M Rso f t h e studiedb i r d s , we foundthatlargerspecies,whichhaveevolvedlargercells(atleastinfivet i s s u e s ) , h aves i m ultaneouslye volvedl owerm a s s -
specificBMRs.Asimilarpatternininterspecificdifferencesinbodymas s,cells i z e a n d standardm e tabolicratesh a s a l s o b e e n f o u n d i n ectothermica n i m a l s , e . g . M a d a g a s carg e c kos( S t a rost ováe t a l . , 2009).Additionally,large-celledtriploidshavelowermass- specificmetabolicratesthandosmall-
celleddiploidsinCobitisfish(Maciaketal.,2011)andPelophylaxfrog s(Hermaniuketal.,2017).Theevidencef oran e gativea s s o c iation b e t weenc e l l s izea nd m a s s -
specificmetabolicrateagreeswiththepredictionoftheTOCSthatabodyb uiltfromlargercellshasarelativelyloweramountofcellmembran esa n d , t h e refore,wastesrelativelyl e s s e n e r g y o n maintaining operationalcellmembranes,i.e.inadesiredphysicalande l e c troche micalstate( C z a r n o l es k i e t a l . , 2 0 1 5 a ; K o z łowskietal.,2003;
Szarski,1983).Savingonmaintenancecostsbyincreasingcellsizewou ldbeadvantageousfororganismsthatfacesupplyli mi t ation.H owever ,l a r g e c e l l s c a n i mp ai r p h ysiologicalactivitybydecreasingthetotal exchangeareaofcellmembranesanddecreasingthediffusionefficiency withincells,butsuchdisadvantagesoflargecellsshouldbeofale sserimportanceforsupply-
limitedorganisms.ThishypothesisoftheTOCSpredictsthatlarg e-
celledorganismshavedecreasedphysiologicalefficiency,especiallyw h e n t h eya rec h a l l e n g e d b y a n i n c reasedm e t a b o l i c demand, e . g . c a u sedb y i n c reasesi n p h ysical( c atabolic)o r biosynthetic(a nabolic)work.Insupportofthishypothesis,acomparativestudyofthero tiferKeratellacochlearis(Gosse,1851)indifferentlakesandalongagradien tofwaterdepthsrevealedthatlargerrotiferst h atc o n s i stedo f l a r g e r c e l l s o c c u p i e d c o o l a n d oxygenatedwaters(Czarnolesk ie t a l . , 2 0 1 5 b).A d d i t i onally,a n experimentalstudyoftherotife rLecaneinermis(Bryce,1892)showedthatlargerrotifershaveana dvantageinfertilityoversmallerrotifersincoldandoxygenatedwatersb utthatsmallrotifersthatconsistedofsmallercellshadsuperiorfertilityinwa rmandoxygen-
deficientconditions(Walczyńskaetal.,2015).Tounderstandthee volutiono f l a r g e r c e l l s i n l a r g ers p e c i e s , f u t u restudiess h o u l d investigatewhetherandwhysupplylimitationsincreasewithbodym assand sh ou l d be ba s e d on a widerangeof bo dy m a s s e s . An in triguingpossibilityisthatlargerspeciesbecomesupplylimitedbec ausetheyareselectedagainstoverinvestinginthenetworkofdistrib utionpathways,whichdeliveroxygenandnutrientstocellsandc o l le ctm e t a b o l i t e s f romc e l l s . Toovercomet h i s l i m i t ation,verteb rateswouldneedtodisproportionallyincreasethevolumesoftheirmainart eriesandthetotalamountofbloodrelativetotheirbodymass,which wouldphysicallyhandicaplargerorganisms.Itisnots u r p r i s i n g t h att h e t o t a l volumeo f b l oodi n a b o d y s c a l e s proportionallyw i t h b o d y m a s s , a n d c o nsequently,l e s s c a p i l l arybloodonaverage perfusesagiventissuevolumeinlargerorganisms(Dawson,2003,2005).
Accordingtoourfourthfinding,hepatocyteshaveundergoneanevolut ionarychangeinsizeintheoppositedirectionthanhavetheotherc e l l t y pes,a n d t h i s p atternwasc o n s i stentlyf o u n d i n t h e studiedbirds andmammals.Earlier,Kozłowskietal.
(2010)foundasimilarpatterninadiversegroupofmammalianspeciesbut notinamphibiansandbirds.Interestingly,Czarnoleskietal.
(2016)studiedc e l l s i z e diff erencesbet weent wo s ub s p e c i esof t he la nd snailCornuaspersum(O.F.Müller,1774)andfoundthatthesizeof
cellsintheirhepatopancreas,theanalogofaliverinvertebrates,fol lowedadifferentpatternthandidthesizesofothercelltypes.Macia ketal.
(2014)postulatedthatthesizeofcellsinatissuecanbefunctionallya s s o c iat edw i t h s u p racellularf u n c t i o n s a n d t h e catabolicversusana bolicactivityofatissue.FollowingtheTOCS,weenvisionthatcells izeismatchedtoabalancebetweenthemetabolicdemandandthesup plyofresourcesinatissue,butthisbalancechangeslocallyinabodya ccordingtothemetabolicactivityofatissueandthelocalcharacteristicsof thesupplysystem.Inf act,b o t h t h e l i veri n vertebratesa n d t h e h e p atopancreasi n mollusksarecharacterizedbyanespeciallyhighleve lofanabolicactivity,whichistoalargeextentdirectedtowardsustain ingthefunctionofothertissuesinthebody.Additionally,theliverappearst obeexceptionallywellsuppliedwithoxygenandresources:bloodreaches t h e liverviaa dualperfusionsystemthat,atl e a stin
mammals,receives∼ 25%o f t h e c a r d iaco u t p u t (Vollmara n dMenger,2009),andhepatocytesareindirectcontactwithhepatic capillaries(Kerr,2010).Finally,independentlyofothercellsinabo dy,h e p atocytesc a n u n d e rgoc h romosomalm u l t i p l i cations,wh icha l t e r s t h e i r s i z e a n d t ranslationalactivity( A n atskayaa n d V inogradov,2007,2010).
Ourworkprovidedcrucialinsightthatcellsizeshouldbegivengreate rc o n s i d e rationa s a n o r g a n i s malp ropertyt h atu n d e r g o es adaptivee volutionaryc h a n g e s a m o n g s p e c i e s . T h i s m a cro-
evolutionaryv i ewi s c o nsistentw i t h e m e rgingc o n c l u s i o nsfro mmolecularresearchth atcellsi zecont rolevolvedto optimize t h e metabolicactivityoftissueandorgansandultimatelytomaximizecellul arfitness(Miettinenetal.,2017).Althoughourdatasuggestthatcellsize, bodymassandmetabolicratescanundergoconcertedevolutionarychanges ,togainabetterunderstandingofthesephenomena,studiesofthecom plexcausallinksamongbodysize,cellsize,physiologicalefficiencyand fitnessareneeded.Ifcellsizeisd e m o nstratedt o affectm a i n t e n ancec o stsa n d o r g a n i s m a l performance,t h e n i t s c o n c e r t e d e volut ionw i t h b o d y m a s s a n d metabolicratesuggeststhatthecellular architectureofthebodyisadjusteda l o n g withm a n y o t hero r g a n i s malt raitst o meetthephysiologicals u p p l y a n d d e m a n d o f a g i venstrategy.T h i s interpretationa d d s an ewpe r s p e c t i vet o vie wson t h eb i o l o gicalsignificanceofcells.
MATERIALSANDMETHODS Animals
WestudiedfivespeciesofGalliformesbirdsandsixspeciesofRodentiam ammals,representingtwodistantlyrelatedordersofendotherms.Eachspe cieswasrepresentedbyfivemales.Thenumberofstudiedanimalswasdictatedby theextremelaboriousnessofthehistologicalandmicroscopicproceduresand thecellsizemeasurements.ThechoiceofGalliformesandRodentiawasmotivated bytheirindependentoriginsanddifferentiationintoawiderangeofspecieswithlargedif ferencesinadultmassbutminimalchangesintheirgeneralbodyplans.Inthisw ay,wemaximizedthestudiedrangeo f b o d y m a s s e s a n d m i n i m i z e d d i f f e rencesi n t h e b i o l o g y o f t h e studieds p e c i e s . Wewerea l s o a b l e t o a d d ressw h e t h e r c o n c e r t e d evolutionaryc h a n g e s i n c e l l s i z e a n d b o d y m a s s o c c u r redi n a s i m i l a r mannerindependentlyinthesetwogr oups.
Allthebirds[commonpheasant,PhasianuscolchicusLinnaeus,1758;chukarpartridg e,Alectorischukar(J.E.Gray,1830);greypartridge,Perdixperdix(Linnaeus,1758);Japanesequ ail,CoturnixjaponicaTemminckandSchlegel,1849;andkingquail,Coturnixchinensi s(Linnaeus,1766)]wereobtainedf romt h e f i e l d stationi n P t a s z kowoo f t h e O sŕodekH o d owliZwierzątŁ ownychi n Parzęczewo-
Cykowo,Poland.TherodentswereobtainedfromdifferentsourcesinPoland[
housemouse,MusmusculusLinnaeus,1758,fromtheJagiellonianCenterofExp erimentalTherapeuticsinKraków;Djungarianhamster,Phodopussungorus(Pallas,
1773),fromtheD e p a r t m e n t o f A n i m a l P h ysiologyo f t h e N i c o l a u s Copernicus
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UniversityinToruń;
brownrat,Rattusnorvegicus(Berkenhout,1769),fromtheDepartmentofPharmacol ogyoftheMedicalCollegeoftheJagiellonianUniversityinKraków;bankvole,Myod esglareolus(Schreber,1780),andcommonvole,Microtusa r v a l i s ( Pallas, 1778),fromtheInstituteofEnvironmentalS c i e n c e s o f t h e J a g i e l l o n i a n U n i versityi n K raków;a n d coypu,Myocastorc o y p u s ( M o l i n a , 1782),fromthePniewyanimalhusbandryfacility].Theanimalsusedinthiss tudywereeuthanizedfollowingtheproceduresoftheinstitutionsfromwhich theanimalswereobtained,whichhadbeenapprovedbytheirlocalethicalcom mittees.Thebirdsandcoypuswereslaughteredaspartofcommercialmeatprodu ction.Otherrodentswereeuthanizedafterlaboratoryexperimentsinwhichthe yservedascontrolgroups.Thedonationofanimalmaterialandallproceduresusedint hisstudyfollowedregulationsofthePolishMinistryofScienceandHigherEducatio n.
Histologicalmethodsandcellsizemeasurements
Priortodissection,animalsweredeprivedoffoodforatleast12handthenweighedto thenearest0.01g(smallrodents),0.1g(birds),or1g(ratsandcoypu).Wetookblo odsamplesfromeachanimalwithheparinizedglasscapillaryt u b e s ( M e d l a b , R a s z y n , Poland)t o p repareb l o o d s m e a r s . ForGalliformes,blood wastakenfromthebrachialvein,andinrodents,fromthecaudalveinorjugularve in(onlycoypus).Bloodsmears weredriedandfixedwithmethanol(Avant orPerformanceMaterialsPolandS.A,Gliwice,Poland)andthenstainedwithGi ll’sIIIHematoxylin(Merck,Darmstadt,Germany)anda1%ethanolsolutio nofEosinY(hereafter,1%EosinY;Analab,Warszawa,Poland)forbirdsorwi th1%EosinYformammals.
Afterremovingfeathersorshavingthecoat,wecollectedaskinsamplefromb etweenthescapulaealongthedorsum.Wedissectedoutthemiddleparto f t h e t r achea,t h e c e n t ralp a r t o f t h e r i g h t l o b e o f t h e l i ver,t h e descendi ngpartoftheduodenumandthewholerightkidney.Thetissuesamplesw erefixedina10%bufferedsolutionofformaldehyde(BioOptica,Milano,Italy).Then,they weredehydratedinethanol(LinegalChemicals,Warszawa,Poland),c l e a redi n S T U l t ra( L e i ca , Wetzlar,G e r m a n y ) a n d embeddedinParaplastPlus(Le ica).Serialsections(4µmthick)werecutwithamotorizedrotarymicrotome(
HyraxM55;Zeiss,Oberkochen,Germany).SlideswerestainedwithGill’sIIIHe matoxylinand1%EosinYandmountedwithCVUltra(Leica).
Erythrocytesin bloo dsmears,t rachealcho nd rocytes,hep atocytesan d
duodenalenterocyteswerephotographedataresolutionof0.033µmperpix elunderalightmicroscope(Eclipse80i;Nikon,Tokyo,Japan)equippedwithac a m e ra(DigitalSight,Nikon)andLuciaMeasurementimageacquisitions o f t ware ( L i m L a b o ratoryI m a g i n g , P raha,C z e c h Republic)usinga 1 0 0 × - m a g n i f i c ationo i l i m m e r s i o n o b j e c t i ve.Cellsf romk i d n eyproximal tubulesandepithelialskincellswerephotographedataresolutionof1µmperpixelus inga40×-
magnificationobjectiveonanautomatizedlightmic roscope(BX5 1 VS; O l y m p u s , Tokyo,Japan )eq u ip ped w it h adigitalcamera(XC10,Olympus)and dotSlide(Olympus)imageacquisitionsoftware.T h e u s e o f t w o m i c roscopics ystems,i n c l u d i n g o n e t h atwasautomated,helpedtoexpeditethedigitizatio nofmicroscopicslidesanddidnotbiasourresultsbecauseweconsistentlyusedthesame systemtoanalyzeagiventissuetypeinallanimals.
Weu s e d i m a g e a n a l ysiss o f t waret o m e a s u rec e l l s : I m a g e J f ro mt h e NationalI n stituteso f H e a l t h ( U S A ) f o r J P E G i m a g e s f rom t h e N i koncameraandcellSensfromOlympusforaspecializedimageformatobtainedf romtheOlympuscamera.Weoutlined60randomlychosenerythrocytespera n imal a n d c a l c u l atedt h e i r areas( µ m2),w h i c h waso u r m e a s u reo f er ythrocytesize.Forbirds,weusedthesamemethodtomeasuretheareasoferythrocytenu clei.Weoutlinedrandomlychosenlacunaeinchondrocytesandcalculatedthe irareas(µm2),whichwasusedasameasureofchondrocytes i z e . I f c h o n d rocyteso c c u r redi n i s o g e n i c g roups,wemeasuredonechondrocyteper group.Cellbordersintheremainingtissueswereoftennotclearlyvisible.Follow ingthemethodsdevelopedbyWieczoreketal.(2015)andCzarnoleskietal.
(2016,2017),wemeasuredtheareasofcellgroupsintissuesamplesfromt heliver,duodenumand kidney(µm2)andthelengthsoflongitudinaltransectsof cellgroupsinskin(µm)samples.Aftercountingthenucleiwithinthemeasuredareasora longeachtransect,wecalculatedtheaveragecellsizebydividingtheareao rtransectlengthbythenumberofnuclei.Tooutlineareasformeasurement,
demarcationlinesbetweenhepatocytesinliversamplesweredrawnequidis tancebetweenneighboringnuclei.Induodenumsamples,weconsidere do n l y e n t e rocytesi n t h e e p i t h e l i a l m u c o u s m e m b ranei n t h e mi ddlepartofvilli.Weusedthebasementmembraneandtheapicalsurfacesofcellsasthelower andupperbordersoflayers,respectively,andtwocellnucleiattwoendsofthelayer asthebeginningandtheendofthelayer.Inkidneysamples,weoutlinedthecross- sectionalareasofproximaltubules(withoutthelumen).Inskinsamples,wecon sideredepithelialcellsofthebasallayer,whichformedlongitudinaltransects.Theend sofatransectweredefinedbytwonuclei,oneateachendofthelineartransectofn uclei.Wemeasuredthefollowingnumberofcellsperindividual‒
birds:52-71intrachea,88-274inlivers,46-257induodenums,332- 521inkidneysand2-
102i n s k i n ; m a m m a l s : 3 7 - 8 9 i n t rachea,7 0 - 2 3 8 i n l i vers,3 7 - 2 9 5 i n
duodenums,9-600inkidneys,13-
166inskin.Finally,wecalculatedtheaveragesizeofeachcelltype(andthe meansizeofnucleiinbirderythrocytes)foreachanimal.Additionally,wec alculatedmeankaryoplasmicratiosforerythrocytesineachbird.
InformationaboutC-values,whereC-valueistheamountofDNAina haploidc e l l ( pg),i n t h e studieds p e c i e s waso b t a i n e d f roma n o n l i n e database(Gregory,2017).IfmorethanoneestimateoftheC- valuewasavailableforaspecies,wecalculatedthemeanC-
value.WefounddataonC-
valueintwospeciesofbirdsandfourspeciesofmammals.Therefore,thedataforbirdswer eusedonlyfordescriptivepurposes,whereasthedataformammalswereanalyzedst atistically.
Respirometryinbirds
Wemeasuredoxygenconsumptionrates(cm3O
2/h)withaparamagnetican alyzerandcarbondioxideproductionrates(cm3C
O2/h)usingthenon- dispersiveinfraredanalysismethod(MAGNOS6GandURAS10E,respect ively,H a r t m a n n a n d B raun,A B B G roup,Z ü r i c h , S w i t z e r l a n d ) . Beforepassingthroughtheanalyzers,whichwereconnectedtoapersonalc omputer,incurrentairwaspassedthroughacolumnofanhydrous calciumchloride(CaCl2;CHEMPUR,PiekaryŚląskie,Poland).Birdsthat underwentm e t a b o l i c m e a s u rementsweret ransferredi n d o o r s f ro mt h e
breedingroom(kingquailandJapanesequail)orfromtheopenaviary(greypartridge,c hukarpartridge,andcommonpheasant)andwereacclimatedtothemeasurementco nditionsfor1h.Birdsweredeprivedoffoodduringthenightbeforethemeasurementsa ndwereweighedpriortorespirometry.Thebirdswereplacedinplasticchamberswi thvolumescloselymatchedtothesizeo f e achb i r d ( 1 . 2 -
2 5 . 0 l).C h a m b e r s w i t h b i r d s werep l acedi n a thermallyinsulated chamberwiththeambienttemperaturecontrolledtothenearest0 . 1 ° C ( E l m e t ro nP T -
2 1 7 d i g i t a l t h e r m o m e t e r , Z a b r z e , Poland).Airflowthroughthe metabolicchamberwasstabilizedwithamassflowmeter(β-
ERG,Warszawa,Poland)at700-
3000ml/min,dependingonthespecies.Therestingmetabolicrateatthermoneutralit y,representingtheBMR,wasdefinedasthelowest10minaveragemetabolicrateofe achbird.Ambienttemperaturesmatchingspecies-
specificthermoneutralzoneswereknownf rome a r l i e r studiesa n d werea s f o l l ows:t h e t e m p e raturewasmaintainedat35.5°Cforkingquail(Pisand Lusnia,2005),at30.5°CforJapanesequail(T.P.,unpublished),at25.0°Cforgre ypartridgeandchukarpartridge(Pis,2003,2010),andat21.0°Cforcommonphe asant(Góreckia n d N owa k , 1 9 9 0 ) . FollowingK l e i b e r ( 1 9 6 1 ) , weu s e d o u r m e a s u redrespiratoryquotientvaluestoexpressmetabolicratesi nmW.Thefollowingenergyequivalentso f1cm3of
O2wereado pted : 20.27 Jforkingquail,
19.95JforJapanesequail,20.08Jforgreypartridge,20.20Jforchukar partridge,and20.01Jforcommonpheasant.
Statisticalanalysis
Weu s e d t h e R S t atisticalPackagef o r t h e statisticala n a l yses( R Devel opmentCoreTeam,2011).The numberofstudiedspecies didnotallo wu s t o achievea s atisfactoryp owerf o r p h y l o g e n e t i c a l l y i n f o r m e d analyses(Garlandetal.,2005).Theanalysisofthekaryoplasmicratiosoferythr
ocyteswasperformedondatafromindividualbirds;otheranalyseswerep e rf o r med o n sp e ci e s m e an s , w h i ch werec al c u l atedf romd atafo rin dividualanimals.Tointegrateinformationoncellsizesindifferenttissue,weperforme daPCAonourc e l l sizedata,separatelyforbirdsandmammals.S co resfo rt h e mo stsig n i fican tPC s weref u r th e r u s ed asou rintegratedmeasures ofcellsizesindifferenttissues.
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Then atureo f P C l o a d i n g s wasu s e d t o exploreh y p o t h e s i s I , w h i c h predictedt h att h e e volutiono f d i f f e rencesi n c e l l s i z e a m o n g s p e c i e s involvedc o o r d i n atedc h a n g e s i n t h e s i z e s o f d i f f e r entc e l l t y p e s . Furthermore,weexaminedw h e t h e r t h i s e volutioni n v olvedc h a n g e s i n genomes i z e / n u c l e u s s i z e . U s i n g i n f o r m ati ono n b i r d e r y t h rocytes,weanalyzedthecorrelationbetweenthesizeofe rythrocytesandtheirnucleiusingagenerallinearmodel(GLM)tocomp arekaryoplasmicratiosforerythrocytesa m o n g s p e c i e s . Wea l s o a n a l y z e d t h e c o r relationb e t weengenomesize(C-
value)andcellsize(thePCscores).TotesthypothesisIIabouttheinvolvement ofchangesincellsizeintheevolutionofbodysize,weexaminedt h e c o r relationb e t weenP C s c o resa n d b o d y m a s s ( l o g10-
transformed).Analyseswereperformedseparatelyforbirdsandmammals.Toexpl oreh y p o t h e s i s I I I , w h i c h p redictst h atl a r g e -
c e l l e d s p e c i e s h aveevolvedlowermass-
specificmetabolicrates,weexaminedthecorrelationbetweenthemass- specificBMRsofbirdsandtheirPCscores.Notethatthisanalysislargelyexplorestheinte gratedeffectsofcellsizeandbodymassif,asp redictedb y h y p o t h e s i s I I , c e l l s i z e a n d b o d y m a s s h avee volvedi n concert.Thehighlyconcertedevolutionofc ellsizeandbodymassmakesitimpossibletoreliablyassesstheindependenteffects ofcellsizeandbodymassonmetabolicrate.Finally,weusedaSMATRprocedure(W artonetal.,2006)tofitastandardizedmajoraxis(SMA)tothelog-
transformeddataforBMRandbodymass.Assumingapowerrelationshipbet weenBMRandbodymass,weusedthistoestimatethemass-
scalingofBMR.Forconsistency,wealsofittedSMAsfortherelationshipsbetweenP Cscoreandlog10bodymassandbetweenmass-specificBMRandPCscore.
Acknowledgements
AnimalswereobtainedcourtesyofB.DydymskiandM.Stokarczak(OśrodekHodowliZwie rzątŁownychinParzęczewo-
Cykowo),E.Kuś(JagiellonianCenterofExperimentalTherapeutics),M.Wojciechowski andM.Jefimow(DepartmentofAnimalPhysiology,NicolausCopernicusUniversity),S.C hłopicki(DepartmentofPharmacology,MedicalCollegeofJagiellonianUniversityandJagi ellonianCenterofExperimentalTherapeutics),P.Koteja,M.KruczekandE.Pochroń(Instit uteofEnvironmentalSciences,JagiellonianUniversity),andM.Biedziak(CoypuHusbandr yinPniewy).WethankK.Adamus-Fiszer,P.Kluska,L.Kuriańska-
Piątek,andE.Szmydforhelpduringthestudy.
Competingi n t e rests
Theauthorsdeclarenocompetingorfinancialinterests.
Authorcontributions
Conceptualization:W.M.K.,J.K.;Methodology:M.C.,A.M.L.,T.P.,W.M.K.,J.K.;Software:
F.K.;Validation:M.C.,A.M.L.,F.K.;Formalanalysis:M.C.;Investigation:A.M.L.,D.D.- K.,T.P.,K.P.;Datacuration:A.M.L.,F.K.;Writing-originaldraft:M.C.;
Writing-review&editing:M.C.,A.M.L.,D.D.-K.,T.P.,K.P.,F.K.,W.M.K.,J.K.;
Supervision:J.K.;Projectadministration:J.K.;Fundingacquisition:J.K.
Funding
TheresearchwassupportedbythePolishMinistryofScientificResearchandInformation Technology[1720/B/P01/2009/36]andfundsfromtheInstituteofEnvironmentalSciences JagiellonianUniversity[DS/BINOZ/INOS/757/2018].ThedevelopmentofTOCSwassupporte dbythePolishNationalScienceCenter[2016/21/B/NZ8/00303].
Supplementaryi n f o r mation
Supplementaryinformationavailableonlineathttp://bio.biologists.org/loo kup/doi/10.1242/bio.029603.supplemental
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