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Clark MS Thorne MAS King M et al (3 more authors) (2018) Life in the intertidal Cellular responses methylation and epigenetics Functional Ecology 32 (8) pp 1982-1994 ISSN 0269-8463

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1982 科|科 wileyonlinelibrarycomjournalfec Functional Ecology 20183219821994

Receivedr ゴザ September ゴグゲゼ科 |科 Acceptedr ゼ February ゴグゲ芦

DOI 1011111365-243513077

R E S E A R C H A R T I C L E

Life in the intertidal Cellular responses methylation and

epigenetics

Melody S Clark1 科|科Michae旭 As Ss Thorne1科|科Miche旭旭e King1科|科He旭en Hipperson2科|科

Joseph Is Hoffman3科|科L旭oyd Ss Peck1

1British Antarctic Surveyp Natura旭 Environment Research Counci旭p Cambridgep UK

2NERC Biomo旭ecu旭ar Ana旭ysis Faci旭ityp Department of Anima旭 and P旭ant Sciencesp University of Sheffie旭dp Sheffie旭dp UK

3Department of Anima旭 Behaviourp University of Bie旭efe旭dp Bie旭efe旭dp Germany

Correspondence

Melody S Clark

Email msclbasacuk

Funding information

Natura旭 Environment Research Counci旭p Grant｠Award Numberr NBAF芦ジゴ

Hand旭ing Editorr Michae旭 Pfrender

Abstractゲs Phenotypic p旭asticity is essentia旭 for the persistence of organisms under changing

environmental conditions but the control of the relevant cellular mechanisms in-

c旭uding which genes are invo旭ved and the regu旭ation of those genes remains un-

c旭ears One way to address this issue is to eva旭uate 旭inks between gene expressionp methy旭ation and phenotype using transp旭antation and common garden experi-ments within genetica旭旭y homogeneous popu旭ationss

ゴs This approach was taken using the Antarctic 旭impet Nacella concinna In this spe-

ciesp two distinct phenotypes are associated with the intertida旭 and subtida旭 zoness The in situ gene expression and methy旭ation profi旭es of intertida旭 and subtida旭 co-

horts were directly compared before and after reciprocal transplantation as well

as after a common garden acc旭imation to aquarium conditions for ゾ monthssザs Expression profi旭es showed significant modu旭ation of ce旭旭u旭ar metabo旭ism to habi-

tat zone with the intertida旭 profi旭e characterised by transcription modu旭es for an-

tioxidant productionp DNA repair and the cytoske旭eton ref旭ecting the need to cope with continua旭旭y f旭uctuating and stressfu旭 conditions inc旭uding wave actionp UV irradiation and desiccation

ジs Transp旭antation had an effect on gene expressions The subtida旭 anima旭s trans-

p旭anted to the intertida旭 zone modified their gene expression patterns towards that of an intertida旭 profi旭es In contrastp many of the antioxidant genes were sti旭旭 differentia旭旭y expressed in the intertida旭 anima旭s severa旭 weeks after transp旭anta-

tion into the re旭ative旭y benign subtida旭 zonesズs Furthermorep a core of genes invo旭ved in antioxidation was sti旭旭 preferentia旭旭y ex-

pressed in intertida旭 anima旭s at the end of the common garden experiments Thusp acc旭imation in an aquarium tank for ゾ months did not comp旭ete旭y erase the inter-tida旭 gene expression profi旭es

葦s Significant methy旭ation differences were measured between intertida旭 and subtida旭 anima旭s from the wi旭d and after transp旭antationp which were reduced on common garden acc旭imations This suggests that epigenetic factors p旭ay an impor-tant ro旭e in physio旭ogica旭 f旭exibi旭ity associated with environmenta旭 niches

This is an open access artic旭e under the terms of the Creative Commons Attribution Licensep which permits usep distribution and reproduction in any mediump provided the origina旭 work is proper旭y citedsｻ ゴグゲ芦 The Authorss Functional Ecology pub旭ished by John Wi旭ey ｹ Sons Ltd on beha旭f of British Eco旭ogica旭 Societys

科架 科 | 科ゲゾ芦ザFunctional EcologyCLARK ET AL

ゲ科 |科INTRODUC TION

Species distributions and fitness traits are significant旭y impacted by both biotic and abiotic factors in their immediate habitat (often

ca旭旭ed genotypeenvironment interactionsｫq howeverp our current understanding of which genes underpin these processes and their regu旭ation remain 旭imited ｪForsmanp ゴグゲズｫs This is further com-

p旭icated by the fact that many species exhibit f旭exib旭e responsesp whereby high旭y variab旭e phenotypes may be expressed in the ab-

sence of genetic differentiations This phenomenon of phenotypic p旭asticity frequent旭y occurs in mo旭旭uscs where the morpho旭ogica旭 characteristics of shell shape and thickness can vary considerably

with environmenta旭 factorss These morpho旭ogica旭 variants ｪoften ca旭旭ed ecotypesｫ can be induced by a variety of abiotic and biotic factors inc旭uding hydrodynamic stressp temperaturep desiccation and predation ｪHar旭eyp Dennyp Machp ｹ Mi旭旭erp ゴググゾｫs Examp旭es in-

clude Littorina striata in which a nodulose form is associated with

wave she旭tered sites and a smooth form dominating wave exposed sites increased shell thickness in Littorina obtusata in response to

the predatory green crab Carcinus maenas and the Antarctic species Nacella concinna and Laternula ellipticap where she旭旭 thickness var-ies with depth and the incidence of ice berg scour respective旭y ｪDe Wo旭fp Backe旭jaup ｹ Verhagenp ゲゾゾ芦q Harper et a旭sp ゴグゲゴq Hoffmanp Peckp Hi旭旭yardp Zieritzp ｹ C旭arkp ゴグゲグq Trusse旭旭 ｹ Nick旭inp ゴググゴｫs In these casesp genetic homogeneity has been demonstrated across phenotypesp but in other species heritab旭e components and genetic sub structuring has been shown within ecotypes and 旭inked to mi-crohabitatss The c旭assic examp旭es of such phenomena inc旭ude the periwinkles Littorina saxitilis and Littorina fabalis (Johannesson amp

Mikhai旭ovap ゴググジq Johannesson ｹ Tatarenkovp ゲゾゾゼｫp whi旭e in other speciesp such as Mytilus there has been a clear demonstration of

cryptic speciation ｪGrantp Cherryp ｹ Lombardp ゴグゲグｫs These studies emphasise the importance of understanding the genetic background of the species under study when examining phenotypic p旭asticitys

The current understanding of the mo旭ecu旭ar basis of phenotypic p旭asticity is 旭imitedp but such information is essentia旭 not on旭y for un-

derstanding how anima旭s function and interact with their immediate environmentp but a旭so for predicting their capacities to cope when that environment changes ｪSomerop ゴグゲグｫs For examp旭ep such genomic ana旭-yses can he旭p decipher the ce旭旭u旭ar mechanisms under旭ying the com-

petition advantage of c旭ose旭y re旭ated species ｪLockwoodp Sandersp ｹ Somerop ゴグゲグｫp dissect the subt旭eties of mu旭tip旭e environmenta旭 stress-

ors ｪChapman et a旭sp ゴグゲゲｫ and the impact of age on the stress response ｪC旭ark et a旭sp ゴグゲザｫs How these expression patterns are modu旭atedp or more important旭y fixedp is unc旭ear in most cases but epigenetics is in-

creasing旭y being revea旭ed as a key factor imp旭icated in gene regu旭ation in the natura旭 environment ｪBossdorfp Richardsp ｹ Pig旭iuccip ゴググ芦ｫs

In this studyp the Antarctic 旭impet N concinna was used to in-

vestigate ce旭旭u旭ar mechanisms underpinning phenotypic p旭asticitys This is one of the most abundant species in the Antarctic marine environment occurring in the intertida旭 zone and subtida旭旭y down to ゲググ m or more ｪPowe旭旭p ゲゾズゲｫs This is not a homing 旭impet ｪWa旭kerp ゲゾゼゴｫ and unti旭 recent旭y two distinct ecotypes were recognisedr an intertidal polaris formp with a ta旭旭erp thicker she旭旭 and a subtida旭 con-

cinna form with a much 旭ighterp f旭atter she旭旭 that is often microscop-

ica旭旭y wsca旭旭opedx due to the grazing by other 旭impets on encrusting endo旭ithic a旭gae ｪNo旭anp ゲゾゾゲｫs Physio旭ogica旭 differences have a旭so been reported between the two ecotypesp inc旭uding to旭erance to experimenta旭 freezingp metabo旭ic response to air exposurep righting abi旭ity under different temperaturesp therma旭 to旭erances and wet tissue mass ｪMor旭eyp C旭arkp ｹ Peckp ゴグゲグq Wa旭旭erp Wor旭andp Conveyp ｹ Barnesp ゴググ葦q Weihe ｹ Abe旭ep ゴググ芦q Data Sゲ and Sゴｫs This spe-

cies is a broadcast spawner with a free swimming p旭anktonic ve旭iger stage that 旭asts ゲゴ months ｪPeckp Heiserp ｹ C旭arkp ゴグゲ葦ｫ and recent popu旭ation genetic ana旭yses using amp旭ified fragment 旭ength po旭y-

morphisms ｪAFLPsｫ showed no evidence of genetic differentiation between the two ecotypes ｪHoffman et a旭sp ゴグゲグｫs Hencep this spe-

cies disp旭ays considerab旭e physio旭ogica旭 and morpho旭ogica旭 p旭asticity against a genetica旭旭y homogeneous background and represents a good candidate for the mo旭ecu旭ar investigation of phenotypic plasticity

The aim of this study was to investigate not on旭y the gene expres-

sion profi旭es underpinning the physio旭ogica旭 f旭exibi旭ity of N concinna to

the intertida旭 and subtida旭 ｪゲズ mｫ zonesp but a旭so to use methy旭ation sensitive amp旭ified po旭ymorphism ｪMSAPｫ ana旭yses to identify whether epigenetic factors may be invo旭ved in the gene expression differencess To achieve thisp two experiments were carried outr a reciproca旭 trans-

p旭ant experiment in the Antarctic and a common garden experiment where collections of intertidal and subtidal animals were held in the

Cambridge aquarium system for ゾ months to ensure fu旭旭 acc旭imation to 旭aboratory conditionss It was expected that there wou旭d be signif-icant differences in the gene expression and methy旭ation profi旭es of intertidal and subtidal animals in the wild and that transplantation to a

different shore zone wou旭d modify theses It was a旭so expected that the extended acc旭imation in a wcommon garden aquariumx wou旭d reset and remove any differences in gene expression and methy旭ation between intertidal and subtidal cohorts

ゴ科 |科MATERIAL S AND METHODS

ゴsゲ科|科Experimenta旭 design

Anima旭s were co旭旭ected at Rothera Research Stationp Ade旭aide Is旭andp Antarctic Peninsu旭a ｪ葦ゼoザジ昼グゼ柱Sp 葦芦oグゼ昼ザグ柱Wｫ during the austra旭

K E Y W O R D S

Antarcticp antioxidantsp cytoske旭etonp g旭utathiony旭ationp methy旭ationsensitive amp旭ified po旭ymorphism ｪMSAPｫp Nacella concinnap pentose shunt pathwayp reactive oxygen species

ゲゾ芦ジ科 |科 科架Functional Ecology CLARK ET AL

summer Intertidal animals were hand collected and subtidal animals

co旭旭ected by SCUBA divers from ゲズ ms The anima旭s produced copi-ous amounts of mucus when detached from rocks during co旭旭ection so they were he旭d in the f旭ow through aquarium at Rothera under ambient sea water conditions (c グﾀCｫ for ゴ days prior to samp旭ing to disperse the mucuss To distinguish the transp旭anted anima旭sp the she旭旭s were painted ye旭旭ow ｪゲゴグ intertida旭 anima旭sｫ and white ｪゲゴグ subtida旭 anima旭sｫs Of thesep 葦グ were returned to their origina旭 habi-tat and 葦グ were transp旭anted to the a旭ternative regime ｪFigure ゲｫs The intertida旭 anima旭s were p旭aced at the shore end of a 旭ong gent旭y s旭oping rocky gu旭旭y ｪc 芦 m 旭ongｫp which then dropped steep旭y into deep waters The subtida旭 anima旭s were p旭aced on 旭arge f旭at rocks at ゲズ m ｪFigure ゴｫs One month 旭aterp two sets of transp旭anted anima旭s

were sampled (each of n ゴジｫs Three sets of contro旭s ｪeach of n ゴジｫ were a旭so samp旭ed from each zoner intertida旭 and subtida旭 anima旭s at time point zerop intertida旭 and subtida旭 anima旭s at ゲ month ｪtime｠seasona旭 contro旭ｫ and painted anima旭s that had been p旭aced back into their origina旭 habitat and samp旭ed at ゲ month ｪmanipu旭ation con-

tro旭sｫ ｪFigure ゲp Tab旭e ゲｫs For each anima旭p the she旭旭 旭engthp height and width were measured with vernier ca旭ipers ｪmmｫs

ゴsゴ科|科Limpet migration

During the experimentp the transp旭anted anima旭s started to move back to their origina旭 habitatq hencep ad hoc observations of the movement of the transp旭anted 旭impets were carried outs Painted

F IGURE ゲ科Representation of both the

transp旭ant and acc旭imation experimenta旭 designs

F IGURE ゴ科 Intertidal and subtidal

environments near Rotheras ｪaｫ Genera旭 view of the intertida旭 region around Rothera baseq ｪbｫ direct view of the gu旭旭y where the 旭impets were transp旭antedp fi旭旭ed with brash iceq ｪcｫ transp旭anted 旭impets in the intertida旭q ｪdｫ transp旭anted 旭impets in the subtida旭 at ゲズ ms Photos courtesy of L旭oyd Peck

(a) (b)

(c) (d)

科架 科 | 科ゲゾ芦ズFunctional EcologyCLARK ET AL

she旭旭 co旭our was noted and anima旭 position 旭ogged either by measur-ing how far they had moved from the origina旭 site they were trans-

p旭anted to or by ranking their position in terms of distance moveds

ゴsザ科|科RNA extraction protoco旭 and sequencing

RNA was extracted from the foot tissue ｪn 葦ｫ for each set of contro旭 and transp旭anted anima旭s using TRI reagent ｪBio旭ineｫ and purified on RNeasy mini co旭umns ｪQiagenｫ according to manufacturersv instruc-

tionss RNA was quantified using a NanoDrop ND ゲグググ ｪLabTech Internationa旭ｫs The individua旭s from each treatment ｪn 葦ｫ were poo旭ed in equa旭 amounts for RNA Seq to produce a sing旭e 旭ibrary for each treatments An initia旭 sequencing runp using two separate libraries of pooled subtidal and intertidal control animals was per-

formed on the Roche ジズジ GS FLX Titanium p旭atform ｪUniversity of Cambridgep Department of Biochemistry Sequencing Faci旭ityｫ to confirm expression differences between the intertida旭 and subtida旭 cohorts before more extensive sequencing of the transp旭ant experi-ments Further sequencing was performed on a旭旭 contro旭 and trans-

p旭ant treatments on an I旭旭umina GAIIx at Edinburgh Genomicss

ゴsジ科|科Transp旭ant experimentr Generation of backbone transcriptome and mapping

The sequences from the origina旭 ジズジ pyrosequencing run ｪゲゲp葦芦芦p芦ジザ readsｫ were assemb旭ed using Newb旭er ｪRocheｫp yie旭ding ゾp芦グゲ contigs with a mean 旭ength of 葦ジ葦 bps These were annotated using the GenBank nr database ｪBensonp Karsch Mizrachip Lipmanp Oste旭旭p ｹ Whee旭erp ゴググゼｫ using a thresho旭d va旭ue for annotation of

any matches below 1eゲグs Two rep旭icate runs from each of the six treatments were run on the GAIIx resu旭ting in ザグ bp sequences of subtida旭 contro旭 time グ ｪSTCｫ ゲゲp葦ゾズpゲゲグq subtida旭 contro旭 ゲ month ｪSTゲMｫ ジpゼゴザpジグゴq subtida旭 painted contro旭 ｪSTSTｫ ズpジゼズpジゾゼq in-

tertida旭 contro旭 time グ ｪITCｫ ジpゼ芦ズpグ芦ゾq intertida旭 contro旭 ゲ month ｪITゲMｫ ザpゾザグpゴ葦ゼq intertida旭 painted contro旭 ｪITITｫ ゲ葦pゾゾゲpゼゼザq intertida旭 transp旭ant ｪITSTｫ ズpジジグp葦ゲ葦q subtida旭 transp旭ant ｪSTITｫ ズpゴグゾpゴゼゴs These were mapped onto the reference contigs using Maq ｪLip Ruanp ｹ Durbinp ゴググ芦ｫs Three approaches were used to determine the significant旭y differentia旭旭y expressed contigs and two of these were app旭ied sequentia旭旭y to the transp旭ant treatments for added stringencys A norma旭ised transcripts per mi旭旭ion ｪtpmｫ va旭ue from the mapping onto the contig was compared to the contro旭 set by dividing the tpm va旭ue to the maximum of the contro旭 set ｪfor STr STIT｠maxｪSTCpSTCゲMpSTSTｫｫs A twofo旭d cut off criteria was re-

quired for se旭ection at this stages The use the counts was used in an-

other approach in a ratio test where the mapping for a transp旭ant setp for examp旭e STITp was tested against each different contro旭 set inde-

pendent旭y ｪises STIT compared to STCp STIT compared to STCゲM and STIT compared to STSTｫ and the p- values adjusted with a selection

cut- off of 01 The final results were the joint selection of these two

criterias The Bayesian mode旭旭ing program BAYSEQ ｪHardcast旭e ｹ Ke旭旭yp ゴグゲグｫ was used using this rep旭icate structure to determine the popu-

lation differences with an adjusted p- value cut- off of 05 Different

cut offs were used according to the approach as the se旭ection cri-teria are independentq the TPM and fo旭d change require stricter p-

va旭ue cut offsp whereas the Bayseq is a旭ready strictp and the cut off va旭ue was chosen to retrieve the most significant resu旭ting geness The final selection was an intersection of these two independent

TABLE ゲ科Exp旭anation of the experimenta旭 regimes and associated samp旭ing

Samples Experimenta旭 va旭ue

Subtidal controls

Subtida旭 anima旭s samp旭ed at time グ ｪSTCｫ Contro旭 at the start of the experiment

Subtida旭 anima旭s samp旭ed at ゲ month ｪSTゲMｫ Contro旭 at the end of the experiment ｪtempora旭 contro旭ｫ

Subtida旭 anima旭sp she旭旭s painted and rep旭aced at ゲズ m ｪSTSTｫ Contro旭 for experimenta旭 manipu旭ation ｪpaintedｫ and a旭so timep as samp旭ed at the end of the experiment ｪtempora旭 and manipu旭ation contro旭ｫ

Intertidal controls

Intertida旭 anima旭s samp旭ed at time グ ｪITCｫ Contro旭 at the start of the experiment

Intertida旭 anima旭s samp旭ed at ゲ month ｪITゲMｫ Contro旭 at the end of the experiment ｪtempora旭 contro旭ｫ

Intertida旭 anima旭sp she旭旭s painted and rep旭aced into the intertida旭 zone ｪITITｫ

Contro旭 for experimenta旭 manipu旭ation ｪpaintedｫ and a旭so timep as samp旭ed at the end of the experiment ｪtempora旭 and manipu旭ation contro旭ｫ

Transplanted animals

Subtida旭 anima旭s transp旭anted to the intertida旭 zone ｪSTITｫ Transplant

Intertida旭 anima旭s transp旭anted to ゲズ m ｪITSTｫ Transplant

9- Month acclimation

Intertida旭 anima旭s acc旭imated in the aquarium for ゾ months ｪITAｫ

Common garden acc旭imation in the aquarium

Subtida旭 anima旭s acc旭imated in the aquarium for ゾ months ｪSTAｫ

Common garden acc旭imation in the aquarium

Sample codes are in brackets

ゲゾ芦葦科 |科 科架Functional Ecology CLARK ET AL

approachess Those upregu旭ated transcriptsp which were putative旭y annotated using B旭ast sequence simi旭arity searching were manua旭旭y verified and then further ana旭ysed using the STRING program to visu-

a旭ise potentia旭 proteinprotein interactions using UniProtKB human identifiers ｪhttpsr｠｠stringdbsorg｠ｫs

ゴsズ科|科Acc旭imation studies

A further set of intertida旭 and subtida旭 anima旭s was co旭旭ected in the aus-

tral summer of 2014 These were transported to the UK and held sepa-

rate旭y in the Cambridge aquarium for ゾ months in a common garden experiments The aquarium is a re circu旭ation system he旭d at グ グsゲﾀCp sa旭inity at ザジ psu ｪゲｫ and a ゲゴrゲゴ 旭ightrdark regimes The anima旭s were he旭d in the same aquarium tank at simi旭ar densitiesp but the subtida旭 and intertida旭 cohorts were kept separateds They fed on a旭gae growing on the aquarium wa旭旭ss There were no morta旭ities during the acc旭imation periods The RNAs from foot tissue were extracted as described above from six simi旭ar sized anima旭s from the intertida旭 ｪshe旭旭 size ゴズsゴ芦 グsザグ mm SE meanｫ and the subtida旭 ｪshe旭旭 size ゴズsジ グsジ芦 mm SE meanｫs There was no significant difference between the she旭旭s sizes of each cohort (T グsゴゲp p sジゲp df 芦ｫs The six RNA extractions from each of the intertidal and subtidal animals were pooled to form two libraries of

subtida旭 and intertida旭 RNAss TruSeqvゴ 旭ibraries were made from each of the two poo旭s and subjected to a MiSeq ゼズ bp paired end read run ｪUniversity of Cambridgep Department of Biochemistry Sequencing Faci旭ityｫs Each 旭ibrary was mapped to the origina旭 transcriptome back-

bone and ana旭ysed as described above se旭ecting significant differentia旭 expression on fo旭d change and p- value adjusted ratio test

ゴs葦科|科Methy旭ation studies

DNA was extracted from the foot tissue of intertida旭 and subtida旭 an-

imals (the controls and transplanted individuals as described above

Figure ゲp Tab旭e ゲｫ using the DNeasy B旭ood and Tissue kit ｪQiagenｫ according to manufacturersv instructionss The DNA concentration and qua旭ity were checked by a spectrophotometer ｪNanoDropp ND ゲグググｫ and an Agi旭ent ゴゴググ TapeStation systems To detect meth-

y旭ation patterns in the different DNA samp旭esp a modified AFLP technique using methy旭ation sensitive restriction enzyme ｪMspI and

HpaIIｫ digestion was carried out ｪData Sザｫs

ザ科 |科RESULTS

ザsゲ科|科Transp旭antationp migration and recapture of transplanted limpets

The aim was to samp旭e ゴジ anima旭s for each treatmentp inc旭uding the contro旭ss This was possib旭e in a旭旭 casesp with the exception of the subtida旭 anima旭s transp旭anted to the intertida旭 zone where it was on旭y possib旭e to retrieve ゲゾ individua旭s from the intertida旭 zones A旭旭 anima旭s were sexu-

a旭旭y maturep a旭though individua旭 sexes were not noteds There was no significant size difference between any of the intertida旭 anima旭s sam-

p旭ed in this experiment ｪone way ANOVA Fザpゾズ グsズゼp p s葦ザ葦ｫs The

subtida旭 anima旭s samp旭ed showed significant differences in size ｪone way ANOVA Fザpゾグ 葦sゲ葦p p sググゲｫs There was no difference between the three sets of subtida旭 contro旭 anima旭s ｪTukey グsグズｫp but the re-

trieved anima旭s transp旭anted to the intertida旭 were significant旭y sma旭旭er than the contro旭s ｪTukey グsグズｫs These transp旭anted anima旭s ｪmean 旭ength ゲゾs芦 mm グsゾ SEｫ and a旭so the contro旭 subtida旭 anima旭sp which were samp旭edp painted and put back into the subtida旭 ｪゴザsゴ mm グs芦 SEｫ ｪdifference in sizer T = 284 p = 0007 df ザゾｫ were from the same origina旭 samp旭e of ゲゴグ painted anima旭s and were a旭旭ocated at random between the two 旭ocationss Given the prob旭ems of retrieving a fu旭旭 set of ゴジ subtida旭 anima旭s from the intertida旭 zone after a monthp the most 旭ike旭y conc旭usion was that the 旭arger anima旭s were either faster or more capab旭e at returning to the subtida旭 zoneq hencep the observed differ-ence in size of the retrieved transp旭anted subtida旭 anima旭ss

This species is not a homing 旭impet ｪWa旭kerp ゲゾゼゴｫq thereforep it was surprising to see that the transp旭anted anima旭s were moving back to their origina旭 habitatss This migration was re旭ative旭y rapid as evi-denced at the fina旭 samp旭ing by the inabi旭ity to retrieve more than ゲゾ subtida旭 individua旭s ｪfrom an origina旭 n 葦グｫ from the intertida旭 gu旭旭yp which inc旭uded samp旭ing in ゲ m of water at 旭ow tides At severa旭 time points during the experimentp the movement of anima旭s was cata-

旭ogueds After ザ daysp ranking of anima旭s in the intertida旭 gu旭旭y showed that the transp旭anted subtida旭 individua旭s had migrated further down the shore towards deeper water than the intertidal limpets (Spearman

rank order [rho] correlation = 1 p sグググゲｫs At ザ weeksp the measure-

ment of distance moved a旭ong the gu旭旭y showed that the subtida旭 anima旭s had moved significant旭y c旭oser to the deeper water than the replaced intertidal animals (t- test T ジsザグq p lt 0001 df ザゴｫs In the subtida旭 at ゲズ mp after ゴ weeks most subtida旭 anima旭s had not moved off the rocks they were rep旭aced ontop but the transp旭anted intertida旭 anima旭s had moved significant旭y towards more sha旭旭ow depths ｪt- test

T = 308 p = 006 df ゲゾｫs In each casep anima旭s that were rep旭aced in their origina旭 habitat did not trave旭 towards the opposite zones

ザsゴ科|科Gene expression ana旭yses from the transp旭ant experiment

To determine if gene expression profi旭es had been modified with trans-

p旭antationp the transcripts upregu旭ated in the transp旭anted anima旭s were compared with contro旭 anima旭s from both their origina旭 environments and a旭so the contro旭 anima旭s from the zone to which they had been trans-

p旭anteds This expression profi旭ing revea旭ed that intertida旭 transp旭ants maintained the wintertida旭 gene profi旭ex despite being transp旭anteds A much bigger change was found with the transp旭anted subtida旭 anima旭sp which indicated that they were re setting their ce旭旭u旭ar machinery to cope with their new stressfu旭 intertida旭 environment ｪTab旭e ゴｫs These shifts in gene expression patterns are described in greater detai旭 be旭ows

ザsザ科|科Intertida旭 transp旭ants compared with intertida旭 contro旭 anima旭s ｪorigina旭 habitatｫ

There was a sma旭旭 difference numerica旭旭y in the expression pro-

fi旭es in this comparisonp with on旭y ゲジ unique annotations in the

ゲゾ芦ジ科 |科 科架Functional Ecology CLARK ET AL

summer Intertidal animals were hand collected and subtidal animals

co旭旭ected by SCUBA divers from ゲズ ms The anima旭s produced copi-ous amounts of mucus when detached from rocks during co旭旭ection so they were he旭d in the f旭ow through aquarium at Rothera under ambient sea water conditions (c グﾀCｫ for ゴ days prior to samp旭ing to disperse the mucuss To distinguish the transp旭anted anima旭sp the she旭旭s were painted ye旭旭ow ｪゲゴグ intertida旭 anima旭sｫ and white ｪゲゴグ subtida旭 anima旭sｫs Of thesep 葦グ were returned to their origina旭 habi-tat and 葦グ were transp旭anted to the a旭ternative regime ｪFigure ゲｫs The intertida旭 anima旭s were p旭aced at the shore end of a 旭ong gent旭y s旭oping rocky gu旭旭y ｪc 芦 m 旭ongｫp which then dropped steep旭y into deep waters The subtida旭 anima旭s were p旭aced on 旭arge f旭at rocks at ゲズ m ｪFigure ゴｫs One month 旭aterp two sets of transp旭anted anima旭s

were sampled (each of n ゴジｫs Three sets of contro旭s ｪeach of n ゴジｫ were a旭so samp旭ed from each zoner intertida旭 and subtida旭 anima旭s at time point zerop intertida旭 and subtida旭 anima旭s at ゲ month ｪtime｠seasona旭 contro旭ｫ and painted anima旭s that had been p旭aced back into their origina旭 habitat and samp旭ed at ゲ month ｪmanipu旭ation con-

tro旭sｫ ｪFigure ゲp Tab旭e ゲｫs For each anima旭p the she旭旭 旭engthp height and width were measured with vernier ca旭ipers ｪmmｫs

ゴsゴ科|科Limpet migration

During the experimentp the transp旭anted anima旭s started to move back to their origina旭 habitatq hencep ad hoc observations of the movement of the transp旭anted 旭impets were carried outs Painted

F IGURE ゲ科Representation of both the

transp旭ant and acc旭imation experimenta旭 designs

F IGURE ゴ科 Intertidal and subtidal

environments near Rotheras ｪaｫ Genera旭 view of the intertida旭 region around Rothera baseq ｪbｫ direct view of the gu旭旭y where the 旭impets were transp旭antedp fi旭旭ed with brash iceq ｪcｫ transp旭anted 旭impets in the intertida旭q ｪdｫ transp旭anted 旭impets in the subtida旭 at ゲズ ms Photos courtesy of L旭oyd Peck

(a) (b)

(c) (d)

科架 科 | 科ゲゾ芦ズFunctional EcologyCLARK ET AL

she旭旭 co旭our was noted and anima旭 position 旭ogged either by measur-ing how far they had moved from the origina旭 site they were trans-

p旭anted to or by ranking their position in terms of distance moveds

ゴsザ科|科RNA extraction protoco旭 and sequencing

RNA was extracted from the foot tissue ｪn 葦ｫ for each set of contro旭 and transp旭anted anima旭s using TRI reagent ｪBio旭ineｫ and purified on RNeasy mini co旭umns ｪQiagenｫ according to manufacturersv instruc-

tionss RNA was quantified using a NanoDrop ND ゲグググ ｪLabTech Internationa旭ｫs The individua旭s from each treatment ｪn 葦ｫ were poo旭ed in equa旭 amounts for RNA Seq to produce a sing旭e 旭ibrary for each treatments An initia旭 sequencing runp using two separate libraries of pooled subtidal and intertidal control animals was per-

formed on the Roche ジズジ GS FLX Titanium p旭atform ｪUniversity of Cambridgep Department of Biochemistry Sequencing Faci旭ityｫ to confirm expression differences between the intertida旭 and subtida旭 cohorts before more extensive sequencing of the transp旭ant experi-ments Further sequencing was performed on a旭旭 contro旭 and trans-

p旭ant treatments on an I旭旭umina GAIIx at Edinburgh Genomicss

ゴsジ科|科Transp旭ant experimentr Generation of backbone transcriptome and mapping

The sequences from the origina旭 ジズジ pyrosequencing run ｪゲゲp葦芦芦p芦ジザ readsｫ were assemb旭ed using Newb旭er ｪRocheｫp yie旭ding ゾp芦グゲ contigs with a mean 旭ength of 葦ジ葦 bps These were annotated using the GenBank nr database ｪBensonp Karsch Mizrachip Lipmanp Oste旭旭p ｹ Whee旭erp ゴググゼｫ using a thresho旭d va旭ue for annotation of

any matches below 1eゲグs Two rep旭icate runs from each of the six treatments were run on the GAIIx resu旭ting in ザグ bp sequences of subtida旭 contro旭 time グ ｪSTCｫ ゲゲp葦ゾズpゲゲグq subtida旭 contro旭 ゲ month ｪSTゲMｫ ジpゼゴザpジグゴq subtida旭 painted contro旭 ｪSTSTｫ ズpジゼズpジゾゼq in-

tertida旭 contro旭 time グ ｪITCｫ ジpゼ芦ズpグ芦ゾq intertida旭 contro旭 ゲ month ｪITゲMｫ ザpゾザグpゴ葦ゼq intertida旭 painted contro旭 ｪITITｫ ゲ葦pゾゾゲpゼゼザq intertida旭 transp旭ant ｪITSTｫ ズpジジグp葦ゲ葦q subtida旭 transp旭ant ｪSTITｫ ズpゴグゾpゴゼゴs These were mapped onto the reference contigs using Maq ｪLip Ruanp ｹ Durbinp ゴググ芦ｫs Three approaches were used to determine the significant旭y differentia旭旭y expressed contigs and two of these were app旭ied sequentia旭旭y to the transp旭ant treatments for added stringencys A norma旭ised transcripts per mi旭旭ion ｪtpmｫ va旭ue from the mapping onto the contig was compared to the contro旭 set by dividing the tpm va旭ue to the maximum of the contro旭 set ｪfor STr STIT｠maxｪSTCpSTCゲMpSTSTｫｫs A twofo旭d cut off criteria was re-

quired for se旭ection at this stages The use the counts was used in an-

other approach in a ratio test where the mapping for a transp旭ant setp for examp旭e STITp was tested against each different contro旭 set inde-

pendent旭y ｪises STIT compared to STCp STIT compared to STCゲM and STIT compared to STSTｫ and the p- values adjusted with a selection

cut- off of 01 The final results were the joint selection of these two

criterias The Bayesian mode旭旭ing program BAYSEQ ｪHardcast旭e ｹ Ke旭旭yp ゴグゲグｫ was used using this rep旭icate structure to determine the popu-

lation differences with an adjusted p- value cut- off of 05 Different

cut offs were used according to the approach as the se旭ection cri-teria are independentq the TPM and fo旭d change require stricter p-

va旭ue cut offsp whereas the Bayseq is a旭ready strictp and the cut off va旭ue was chosen to retrieve the most significant resu旭ting geness The final selection was an intersection of these two independent

TABLE ゲ科Exp旭anation of the experimenta旭 regimes and associated samp旭ing

Samples Experimenta旭 va旭ue

Subtidal controls

Subtida旭 anima旭s samp旭ed at time グ ｪSTCｫ Contro旭 at the start of the experiment

Subtida旭 anima旭s samp旭ed at ゲ month ｪSTゲMｫ Contro旭 at the end of the experiment ｪtempora旭 contro旭ｫ

Subtida旭 anima旭sp she旭旭s painted and rep旭aced at ゲズ m ｪSTSTｫ Contro旭 for experimenta旭 manipu旭ation ｪpaintedｫ and a旭so timep as samp旭ed at the end of the experiment ｪtempora旭 and manipu旭ation contro旭ｫ

Intertidal controls

Intertida旭 anima旭s samp旭ed at time グ ｪITCｫ Contro旭 at the start of the experiment

Intertida旭 anima旭s samp旭ed at ゲ month ｪITゲMｫ Contro旭 at the end of the experiment ｪtempora旭 contro旭ｫ

Intertida旭 anima旭sp she旭旭s painted and rep旭aced into the intertida旭 zone ｪITITｫ

Contro旭 for experimenta旭 manipu旭ation ｪpaintedｫ and a旭so timep as samp旭ed at the end of the experiment ｪtempora旭 and manipu旭ation contro旭ｫ

Transplanted animals

Subtida旭 anima旭s transp旭anted to the intertida旭 zone ｪSTITｫ Transplant

Intertida旭 anima旭s transp旭anted to ゲズ m ｪITSTｫ Transplant

9- Month acclimation

Intertida旭 anima旭s acc旭imated in the aquarium for ゾ months ｪITAｫ

Common garden acc旭imation in the aquarium

Subtida旭 anima旭s acc旭imated in the aquarium for ゾ months ｪSTAｫ

Common garden acc旭imation in the aquarium

Sample codes are in brackets

ゲゾ芦葦科 |科 科架Functional Ecology CLARK ET AL

approachess Those upregu旭ated transcriptsp which were putative旭y annotated using B旭ast sequence simi旭arity searching were manua旭旭y verified and then further ana旭ysed using the STRING program to visu-

a旭ise potentia旭 proteinprotein interactions using UniProtKB human identifiers ｪhttpsr｠｠stringdbsorg｠ｫs

ゴsズ科|科Acc旭imation studies

A further set of intertida旭 and subtida旭 anima旭s was co旭旭ected in the aus-

tral summer of 2014 These were transported to the UK and held sepa-

rate旭y in the Cambridge aquarium for ゾ months in a common garden experiments The aquarium is a re circu旭ation system he旭d at グ グsゲﾀCp sa旭inity at ザジ psu ｪゲｫ and a ゲゴrゲゴ 旭ightrdark regimes The anima旭s were he旭d in the same aquarium tank at simi旭ar densitiesp but the subtida旭 and intertida旭 cohorts were kept separateds They fed on a旭gae growing on the aquarium wa旭旭ss There were no morta旭ities during the acc旭imation periods The RNAs from foot tissue were extracted as described above from six simi旭ar sized anima旭s from the intertida旭 ｪshe旭旭 size ゴズsゴ芦 グsザグ mm SE meanｫ and the subtida旭 ｪshe旭旭 size ゴズsジ グsジ芦 mm SE meanｫs There was no significant difference between the she旭旭s sizes of each cohort (T グsゴゲp p sジゲp df 芦ｫs The six RNA extractions from each of the intertidal and subtidal animals were pooled to form two libraries of

subtida旭 and intertida旭 RNAss TruSeqvゴ 旭ibraries were made from each of the two poo旭s and subjected to a MiSeq ゼズ bp paired end read run ｪUniversity of Cambridgep Department of Biochemistry Sequencing Faci旭ityｫs Each 旭ibrary was mapped to the origina旭 transcriptome back-

bone and ana旭ysed as described above se旭ecting significant differentia旭 expression on fo旭d change and p- value adjusted ratio test

ゴs葦科|科Methy旭ation studies

DNA was extracted from the foot tissue of intertida旭 and subtida旭 an-

imals (the controls and transplanted individuals as described above

Figure ゲp Tab旭e ゲｫ using the DNeasy B旭ood and Tissue kit ｪQiagenｫ according to manufacturersv instructionss The DNA concentration and qua旭ity were checked by a spectrophotometer ｪNanoDropp ND ゲグググｫ and an Agi旭ent ゴゴググ TapeStation systems To detect meth-

y旭ation patterns in the different DNA samp旭esp a modified AFLP technique using methy旭ation sensitive restriction enzyme ｪMspI and

HpaIIｫ digestion was carried out ｪData Sザｫs

ザ科 |科RESULTS

ザsゲ科|科Transp旭antationp migration and recapture of transplanted limpets

The aim was to samp旭e ゴジ anima旭s for each treatmentp inc旭uding the contro旭ss This was possib旭e in a旭旭 casesp with the exception of the subtida旭 anima旭s transp旭anted to the intertida旭 zone where it was on旭y possib旭e to retrieve ゲゾ individua旭s from the intertida旭 zones A旭旭 anima旭s were sexu-

a旭旭y maturep a旭though individua旭 sexes were not noteds There was no significant size difference between any of the intertida旭 anima旭s sam-

p旭ed in this experiment ｪone way ANOVA Fザpゾズ グsズゼp p s葦ザ葦ｫs The

subtida旭 anima旭s samp旭ed showed significant differences in size ｪone way ANOVA Fザpゾグ 葦sゲ葦p p sググゲｫs There was no difference between the three sets of subtida旭 contro旭 anima旭s ｪTukey グsグズｫp but the re-

trieved anima旭s transp旭anted to the intertida旭 were significant旭y sma旭旭er than the contro旭s ｪTukey グsグズｫs These transp旭anted anima旭s ｪmean 旭ength ゲゾs芦 mm グsゾ SEｫ and a旭so the contro旭 subtida旭 anima旭sp which were samp旭edp painted and put back into the subtida旭 ｪゴザsゴ mm グs芦 SEｫ ｪdifference in sizer T = 284 p = 0007 df ザゾｫ were from the same origina旭 samp旭e of ゲゴグ painted anima旭s and were a旭旭ocated at random between the two 旭ocationss Given the prob旭ems of retrieving a fu旭旭 set of ゴジ subtida旭 anima旭s from the intertida旭 zone after a monthp the most 旭ike旭y conc旭usion was that the 旭arger anima旭s were either faster or more capab旭e at returning to the subtida旭 zoneq hencep the observed differ-ence in size of the retrieved transp旭anted subtida旭 anima旭ss

This species is not a homing 旭impet ｪWa旭kerp ゲゾゼゴｫq thereforep it was surprising to see that the transp旭anted anima旭s were moving back to their origina旭 habitatss This migration was re旭ative旭y rapid as evi-denced at the fina旭 samp旭ing by the inabi旭ity to retrieve more than ゲゾ subtida旭 individua旭s ｪfrom an origina旭 n 葦グｫ from the intertida旭 gu旭旭yp which inc旭uded samp旭ing in ゲ m of water at 旭ow tides At severa旭 time points during the experimentp the movement of anima旭s was cata-

旭ogueds After ザ daysp ranking of anima旭s in the intertida旭 gu旭旭y showed that the transp旭anted subtida旭 individua旭s had migrated further down the shore towards deeper water than the intertidal limpets (Spearman

rank order [rho] correlation = 1 p sグググゲｫs At ザ weeksp the measure-

ment of distance moved a旭ong the gu旭旭y showed that the subtida旭 anima旭s had moved significant旭y c旭oser to the deeper water than the replaced intertidal animals (t- test T ジsザグq p lt 0001 df ザゴｫs In the subtida旭 at ゲズ mp after ゴ weeks most subtida旭 anima旭s had not moved off the rocks they were rep旭aced ontop but the transp旭anted intertida旭 anima旭s had moved significant旭y towards more sha旭旭ow depths ｪt- test

T = 308 p = 006 df ゲゾｫs In each casep anima旭s that were rep旭aced in their origina旭 habitat did not trave旭 towards the opposite zones

ザsゴ科|科Gene expression ana旭yses from the transp旭ant experiment

To determine if gene expression profi旭es had been modified with trans-

p旭antationp the transcripts upregu旭ated in the transp旭anted anima旭s were compared with contro旭 anima旭s from both their origina旭 environments and a旭so the contro旭 anima旭s from the zone to which they had been trans-

p旭anteds This expression profi旭ing revea旭ed that intertida旭 transp旭ants maintained the wintertida旭 gene profi旭ex despite being transp旭anteds A much bigger change was found with the transp旭anted subtida旭 anima旭sp which indicated that they were re setting their ce旭旭u旭ar machinery to cope with their new stressfu旭 intertida旭 environment ｪTab旭e ゴｫs These shifts in gene expression patterns are described in greater detai旭 be旭ows

ザsザ科|科Intertida旭 transp旭ants compared with intertida旭 contro旭 anima旭s ｪorigina旭 habitatｫ

There was a sma旭旭 difference numerica旭旭y in the expression pro-

fi旭es in this comparisonp with on旭y ゲジ unique annotations in the

科架 科 | 科ゲゾ芦ズFunctional EcologyCLARK ET AL

she旭旭 co旭our was noted and anima旭 position 旭ogged either by measur-ing how far they had moved from the origina旭 site they were trans-

p旭anted to or by ranking their position in terms of distance moveds

ゴsザ科|科RNA extraction protoco旭 and sequencing

RNA was extracted from the foot tissue ｪn 葦ｫ for each set of contro旭 and transp旭anted anima旭s using TRI reagent ｪBio旭ineｫ and purified on RNeasy mini co旭umns ｪQiagenｫ according to manufacturersv instruc-

tionss RNA was quantified using a NanoDrop ND ゲグググ ｪLabTech Internationa旭ｫs The individua旭s from each treatment ｪn 葦ｫ were poo旭ed in equa旭 amounts for RNA Seq to produce a sing旭e 旭ibrary for each treatments An initia旭 sequencing runp using two separate libraries of pooled subtidal and intertidal control animals was per-

formed on the Roche ジズジ GS FLX Titanium p旭atform ｪUniversity of Cambridgep Department of Biochemistry Sequencing Faci旭ityｫ to confirm expression differences between the intertida旭 and subtida旭 cohorts before more extensive sequencing of the transp旭ant experi-ments Further sequencing was performed on a旭旭 contro旭 and trans-

p旭ant treatments on an I旭旭umina GAIIx at Edinburgh Genomicss

ゴsジ科|科Transp旭ant experimentr Generation of backbone transcriptome and mapping

The sequences from the origina旭 ジズジ pyrosequencing run ｪゲゲp葦芦芦p芦ジザ readsｫ were assemb旭ed using Newb旭er ｪRocheｫp yie旭ding ゾp芦グゲ contigs with a mean 旭ength of 葦ジ葦 bps These were annotated using the GenBank nr database ｪBensonp Karsch Mizrachip Lipmanp Oste旭旭p ｹ Whee旭erp ゴググゼｫ using a thresho旭d va旭ue for annotation of

any matches below 1eゲグs Two rep旭icate runs from each of the six treatments were run on the GAIIx resu旭ting in ザグ bp sequences of subtida旭 contro旭 time グ ｪSTCｫ ゲゲp葦ゾズpゲゲグq subtida旭 contro旭 ゲ month ｪSTゲMｫ ジpゼゴザpジグゴq subtida旭 painted contro旭 ｪSTSTｫ ズpジゼズpジゾゼq in-

tertida旭 contro旭 time グ ｪITCｫ ジpゼ芦ズpグ芦ゾq intertida旭 contro旭 ゲ month ｪITゲMｫ ザpゾザグpゴ葦ゼq intertida旭 painted contro旭 ｪITITｫ ゲ葦pゾゾゲpゼゼザq intertida旭 transp旭ant ｪITSTｫ ズpジジグp葦ゲ葦q subtida旭 transp旭ant ｪSTITｫ ズpゴグゾpゴゼゴs These were mapped onto the reference contigs using Maq ｪLip Ruanp ｹ Durbinp ゴググ芦ｫs Three approaches were used to determine the significant旭y differentia旭旭y expressed contigs and two of these were app旭ied sequentia旭旭y to the transp旭ant treatments for added stringencys A norma旭ised transcripts per mi旭旭ion ｪtpmｫ va旭ue from the mapping onto the contig was compared to the contro旭 set by dividing the tpm va旭ue to the maximum of the contro旭 set ｪfor STr STIT｠maxｪSTCpSTCゲMpSTSTｫｫs A twofo旭d cut off criteria was re-

quired for se旭ection at this stages The use the counts was used in an-

other approach in a ratio test where the mapping for a transp旭ant setp for examp旭e STITp was tested against each different contro旭 set inde-

pendent旭y ｪises STIT compared to STCp STIT compared to STCゲM and STIT compared to STSTｫ and the p- values adjusted with a selection

cut- off of 01 The final results were the joint selection of these two

criterias The Bayesian mode旭旭ing program BAYSEQ ｪHardcast旭e ｹ Ke旭旭yp ゴグゲグｫ was used using this rep旭icate structure to determine the popu-

lation differences with an adjusted p- value cut- off of 05 Different

cut offs were used according to the approach as the se旭ection cri-teria are independentq the TPM and fo旭d change require stricter p-

va旭ue cut offsp whereas the Bayseq is a旭ready strictp and the cut off va旭ue was chosen to retrieve the most significant resu旭ting geness The final selection was an intersection of these two independent

TABLE ゲ科Exp旭anation of the experimenta旭 regimes and associated samp旭ing

Samples Experimenta旭 va旭ue

Subtidal controls

Subtida旭 anima旭s samp旭ed at time グ ｪSTCｫ Contro旭 at the start of the experiment

Subtida旭 anima旭s samp旭ed at ゲ month ｪSTゲMｫ Contro旭 at the end of the experiment ｪtempora旭 contro旭ｫ

Subtida旭 anima旭sp she旭旭s painted and rep旭aced at ゲズ m ｪSTSTｫ Contro旭 for experimenta旭 manipu旭ation ｪpaintedｫ and a旭so timep as samp旭ed at the end of the experiment ｪtempora旭 and manipu旭ation contro旭ｫ

Intertidal controls

Intertida旭 anima旭s samp旭ed at time グ ｪITCｫ Contro旭 at the start of the experiment

Intertida旭 anima旭s samp旭ed at ゲ month ｪITゲMｫ Contro旭 at the end of the experiment ｪtempora旭 contro旭ｫ

Intertida旭 anima旭sp she旭旭s painted and rep旭aced into the intertida旭 zone ｪITITｫ

Contro旭 for experimenta旭 manipu旭ation ｪpaintedｫ and a旭so timep as samp旭ed at the end of the experiment ｪtempora旭 and manipu旭ation contro旭ｫ

Transplanted animals

Subtida旭 anima旭s transp旭anted to the intertida旭 zone ｪSTITｫ Transplant

Intertida旭 anima旭s transp旭anted to ゲズ m ｪITSTｫ Transplant

9- Month acclimation

Intertida旭 anima旭s acc旭imated in the aquarium for ゾ months ｪITAｫ

Common garden acc旭imation in the aquarium

Subtida旭 anima旭s acc旭imated in the aquarium for ゾ months ｪSTAｫ

Common garden acc旭imation in the aquarium

Sample codes are in brackets

ゲゾ芦葦科 |科 科架Functional Ecology CLARK ET AL

approachess Those upregu旭ated transcriptsp which were putative旭y annotated using B旭ast sequence simi旭arity searching were manua旭旭y verified and then further ana旭ysed using the STRING program to visu-

a旭ise potentia旭 proteinprotein interactions using UniProtKB human identifiers ｪhttpsr｠｠stringdbsorg｠ｫs

ゴsズ科|科Acc旭imation studies

A further set of intertida旭 and subtida旭 anima旭s was co旭旭ected in the aus-

tral summer of 2014 These were transported to the UK and held sepa-

rate旭y in the Cambridge aquarium for ゾ months in a common garden experiments The aquarium is a re circu旭ation system he旭d at グ グsゲﾀCp sa旭inity at ザジ psu ｪゲｫ and a ゲゴrゲゴ 旭ightrdark regimes The anima旭s were he旭d in the same aquarium tank at simi旭ar densitiesp but the subtida旭 and intertida旭 cohorts were kept separateds They fed on a旭gae growing on the aquarium wa旭旭ss There were no morta旭ities during the acc旭imation periods The RNAs from foot tissue were extracted as described above from six simi旭ar sized anima旭s from the intertida旭 ｪshe旭旭 size ゴズsゴ芦 グsザグ mm SE meanｫ and the subtida旭 ｪshe旭旭 size ゴズsジ グsジ芦 mm SE meanｫs There was no significant difference between the she旭旭s sizes of each cohort (T グsゴゲp p sジゲp df 芦ｫs The six RNA extractions from each of the intertidal and subtidal animals were pooled to form two libraries of

subtida旭 and intertida旭 RNAss TruSeqvゴ 旭ibraries were made from each of the two poo旭s and subjected to a MiSeq ゼズ bp paired end read run ｪUniversity of Cambridgep Department of Biochemistry Sequencing Faci旭ityｫs Each 旭ibrary was mapped to the origina旭 transcriptome back-

bone and ana旭ysed as described above se旭ecting significant differentia旭 expression on fo旭d change and p- value adjusted ratio test

ゴs葦科|科Methy旭ation studies

DNA was extracted from the foot tissue of intertida旭 and subtida旭 an-

imals (the controls and transplanted individuals as described above

Figure ゲp Tab旭e ゲｫ using the DNeasy B旭ood and Tissue kit ｪQiagenｫ according to manufacturersv instructionss The DNA concentration and qua旭ity were checked by a spectrophotometer ｪNanoDropp ND ゲグググｫ and an Agi旭ent ゴゴググ TapeStation systems To detect meth-

y旭ation patterns in the different DNA samp旭esp a modified AFLP technique using methy旭ation sensitive restriction enzyme ｪMspI and

HpaIIｫ digestion was carried out ｪData Sザｫs

ザ科 |科RESULTS

ザsゲ科|科Transp旭antationp migration and recapture of transplanted limpets

The aim was to samp旭e ゴジ anima旭s for each treatmentp inc旭uding the contro旭ss This was possib旭e in a旭旭 casesp with the exception of the subtida旭 anima旭s transp旭anted to the intertida旭 zone where it was on旭y possib旭e to retrieve ゲゾ individua旭s from the intertida旭 zones A旭旭 anima旭s were sexu-

a旭旭y maturep a旭though individua旭 sexes were not noteds There was no significant size difference between any of the intertida旭 anima旭s sam-

p旭ed in this experiment ｪone way ANOVA Fザpゾズ グsズゼp p s葦ザ葦ｫs The

subtida旭 anima旭s samp旭ed showed significant differences in size ｪone way ANOVA Fザpゾグ 葦sゲ葦p p sググゲｫs There was no difference between the three sets of subtida旭 contro旭 anima旭s ｪTukey グsグズｫp but the re-

trieved anima旭s transp旭anted to the intertida旭 were significant旭y sma旭旭er than the contro旭s ｪTukey グsグズｫs These transp旭anted anima旭s ｪmean 旭ength ゲゾs芦 mm グsゾ SEｫ and a旭so the contro旭 subtida旭 anima旭sp which were samp旭edp painted and put back into the subtida旭 ｪゴザsゴ mm グs芦 SEｫ ｪdifference in sizer T = 284 p = 0007 df ザゾｫ were from the same origina旭 samp旭e of ゲゴグ painted anima旭s and were a旭旭ocated at random between the two 旭ocationss Given the prob旭ems of retrieving a fu旭旭 set of ゴジ subtida旭 anima旭s from the intertida旭 zone after a monthp the most 旭ike旭y conc旭usion was that the 旭arger anima旭s were either faster or more capab旭e at returning to the subtida旭 zoneq hencep the observed differ-ence in size of the retrieved transp旭anted subtida旭 anima旭ss

This species is not a homing 旭impet ｪWa旭kerp ゲゾゼゴｫq thereforep it was surprising to see that the transp旭anted anima旭s were moving back to their origina旭 habitatss This migration was re旭ative旭y rapid as evi-denced at the fina旭 samp旭ing by the inabi旭ity to retrieve more than ゲゾ subtida旭 individua旭s ｪfrom an origina旭 n 葦グｫ from the intertida旭 gu旭旭yp which inc旭uded samp旭ing in ゲ m of water at 旭ow tides At severa旭 time points during the experimentp the movement of anima旭s was cata-

旭ogueds After ザ daysp ranking of anima旭s in the intertida旭 gu旭旭y showed that the transp旭anted subtida旭 individua旭s had migrated further down the shore towards deeper water than the intertidal limpets (Spearman

rank order [rho] correlation = 1 p sグググゲｫs At ザ weeksp the measure-

ment of distance moved a旭ong the gu旭旭y showed that the subtida旭 anima旭s had moved significant旭y c旭oser to the deeper water than the replaced intertidal animals (t- test T ジsザグq p lt 0001 df ザゴｫs In the subtida旭 at ゲズ mp after ゴ weeks most subtida旭 anima旭s had not moved off the rocks they were rep旭aced ontop but the transp旭anted intertida旭 anima旭s had moved significant旭y towards more sha旭旭ow depths ｪt- test

T = 308 p = 006 df ゲゾｫs In each casep anima旭s that were rep旭aced in their origina旭 habitat did not trave旭 towards the opposite zones

ザsゴ科|科Gene expression ana旭yses from the transp旭ant experiment

To determine if gene expression profi旭es had been modified with trans-

p旭antationp the transcripts upregu旭ated in the transp旭anted anima旭s were compared with contro旭 anima旭s from both their origina旭 environments and a旭so the contro旭 anima旭s from the zone to which they had been trans-

p旭anteds This expression profi旭ing revea旭ed that intertida旭 transp旭ants maintained the wintertida旭 gene profi旭ex despite being transp旭anteds A much bigger change was found with the transp旭anted subtida旭 anima旭sp which indicated that they were re setting their ce旭旭u旭ar machinery to cope with their new stressfu旭 intertida旭 environment ｪTab旭e ゴｫs These shifts in gene expression patterns are described in greater detai旭 be旭ows

ザsザ科|科Intertida旭 transp旭ants compared with intertida旭 contro旭 anima旭s ｪorigina旭 habitatｫ

There was a sma旭旭 difference numerica旭旭y in the expression pro-

fi旭es in this comparisonp with on旭y ゲジ unique annotations in the

ゲゾ芦葦科 |科 科架Functional Ecology CLARK ET AL

approachess Those upregu旭ated transcriptsp which were putative旭y annotated using B旭ast sequence simi旭arity searching were manua旭旭y verified and then further ana旭ysed using the STRING program to visu-

a旭ise potentia旭 proteinprotein interactions using UniProtKB human identifiers ｪhttpsr｠｠stringdbsorg｠ｫs

ゴsズ科|科Acc旭imation studies

A further set of intertida旭 and subtida旭 anima旭s was co旭旭ected in the aus-

tral summer of 2014 These were transported to the UK and held sepa-

rate旭y in the Cambridge aquarium for ゾ months in a common garden experiments The aquarium is a re circu旭ation system he旭d at グ グsゲﾀCp sa旭inity at ザジ psu ｪゲｫ and a ゲゴrゲゴ 旭ightrdark regimes The anima旭s were he旭d in the same aquarium tank at simi旭ar densitiesp but the subtida旭 and intertida旭 cohorts were kept separateds They fed on a旭gae growing on the aquarium wa旭旭ss There were no morta旭ities during the acc旭imation periods The RNAs from foot tissue were extracted as described above from six simi旭ar sized anima旭s from the intertida旭 ｪshe旭旭 size ゴズsゴ芦 グsザグ mm SE meanｫ and the subtida旭 ｪshe旭旭 size ゴズsジ グsジ芦 mm SE meanｫs There was no significant difference between the she旭旭s sizes of each cohort (T グsゴゲp p sジゲp df 芦ｫs The six RNA extractions from each of the intertidal and subtidal animals were pooled to form two libraries of

subtida旭 and intertida旭 RNAss TruSeqvゴ 旭ibraries were made from each of the two poo旭s and subjected to a MiSeq ゼズ bp paired end read run ｪUniversity of Cambridgep Department of Biochemistry Sequencing Faci旭ityｫs Each 旭ibrary was mapped to the origina旭 transcriptome back-

bone and ana旭ysed as described above se旭ecting significant differentia旭 expression on fo旭d change and p- value adjusted ratio test

ゴs葦科|科Methy旭ation studies

DNA was extracted from the foot tissue of intertida旭 and subtida旭 an-

imals (the controls and transplanted individuals as described above

Figure ゲp Tab旭e ゲｫ using the DNeasy B旭ood and Tissue kit ｪQiagenｫ according to manufacturersv instructionss The DNA concentration and qua旭ity were checked by a spectrophotometer ｪNanoDropp ND ゲグググｫ and an Agi旭ent ゴゴググ TapeStation systems To detect meth-

y旭ation patterns in the different DNA samp旭esp a modified AFLP technique using methy旭ation sensitive restriction enzyme ｪMspI and

HpaIIｫ digestion was carried out ｪData Sザｫs

ザ科 |科RESULTS

ザsゲ科|科Transp旭antationp migration and recapture of transplanted limpets

The aim was to samp旭e ゴジ anima旭s for each treatmentp inc旭uding the contro旭ss This was possib旭e in a旭旭 casesp with the exception of the subtida旭 anima旭s transp旭anted to the intertida旭 zone where it was on旭y possib旭e to retrieve ゲゾ individua旭s from the intertida旭 zones A旭旭 anima旭s were sexu-

a旭旭y maturep a旭though individua旭 sexes were not noteds There was no significant size difference between any of the intertida旭 anima旭s sam-

p旭ed in this experiment ｪone way ANOVA Fザpゾズ グsズゼp p s葦ザ葦ｫs The

subtida旭 anima旭s samp旭ed showed significant differences in size ｪone way ANOVA Fザpゾグ 葦sゲ葦p p sググゲｫs There was no difference between the three sets of subtida旭 contro旭 anima旭s ｪTukey グsグズｫp but the re-

trieved anima旭s transp旭anted to the intertida旭 were significant旭y sma旭旭er than the contro旭s ｪTukey グsグズｫs These transp旭anted anima旭s ｪmean 旭ength ゲゾs芦 mm グsゾ SEｫ and a旭so the contro旭 subtida旭 anima旭sp which were samp旭edp painted and put back into the subtida旭 ｪゴザsゴ mm グs芦 SEｫ ｪdifference in sizer T = 284 p = 0007 df ザゾｫ were from the same origina旭 samp旭e of ゲゴグ painted anima旭s and were a旭旭ocated at random between the two 旭ocationss Given the prob旭ems of retrieving a fu旭旭 set of ゴジ subtida旭 anima旭s from the intertida旭 zone after a monthp the most 旭ike旭y conc旭usion was that the 旭arger anima旭s were either faster or more capab旭e at returning to the subtida旭 zoneq hencep the observed differ-ence in size of the retrieved transp旭anted subtida旭 anima旭ss

This species is not a homing 旭impet ｪWa旭kerp ゲゾゼゴｫq thereforep it was surprising to see that the transp旭anted anima旭s were moving back to their origina旭 habitatss This migration was re旭ative旭y rapid as evi-denced at the fina旭 samp旭ing by the inabi旭ity to retrieve more than ゲゾ subtida旭 individua旭s ｪfrom an origina旭 n 葦グｫ from the intertida旭 gu旭旭yp which inc旭uded samp旭ing in ゲ m of water at 旭ow tides At severa旭 time points during the experimentp the movement of anima旭s was cata-

旭ogueds After ザ daysp ranking of anima旭s in the intertida旭 gu旭旭y showed that the transp旭anted subtida旭 individua旭s had migrated further down the shore towards deeper water than the intertidal limpets (Spearman

rank order [rho] correlation = 1 p sグググゲｫs At ザ weeksp the measure-

ment of distance moved a旭ong the gu旭旭y showed that the subtida旭 anima旭s had moved significant旭y c旭oser to the deeper water than the replaced intertidal animals (t- test T ジsザグq p lt 0001 df ザゴｫs In the subtida旭 at ゲズ mp after ゴ weeks most subtida旭 anima旭s had not moved off the rocks they were rep旭aced ontop but the transp旭anted intertida旭 anima旭s had moved significant旭y towards more sha旭旭ow depths ｪt- test

T = 308 p = 006 df ゲゾｫs In each casep anima旭s that were rep旭aced in their origina旭 habitat did not trave旭 towards the opposite zones

ザsゴ科|科Gene expression ana旭yses from the transp旭ant experiment

To determine if gene expression profi旭es had been modified with trans-

p旭antationp the transcripts upregu旭ated in the transp旭anted anima旭s were compared with contro旭 anima旭s from both their origina旭 environments and a旭so the contro旭 anima旭s from the zone to which they had been trans-

p旭anteds This expression profi旭ing revea旭ed that intertida旭 transp旭ants maintained the wintertida旭 gene profi旭ex despite being transp旭anteds A much bigger change was found with the transp旭anted subtida旭 anima旭sp which indicated that they were re setting their ce旭旭u旭ar machinery to cope with their new stressfu旭 intertida旭 environment ｪTab旭e ゴｫs These shifts in gene expression patterns are described in greater detai旭 be旭ows

ザsザ科|科Intertida旭 transp旭ants compared with intertida旭 contro旭 anima旭s ｪorigina旭 habitatｫ

There was a sma旭旭 difference numerica旭旭y in the expression pro-

fi旭es in this comparisonp with on旭y ゲジ unique annotations in the

科架 科 | 科ゲゾ芦ゼFunctional EcologyCLARK ET AL

differentia旭旭y expressed transcripts ｪTab旭e ゴｫp that is the intertida旭 transp旭ants maintained a very simi旭ar profi旭e to that of the origina旭 intertidal controls

ザsジ科|科Intertida旭 transp旭ants compared with subtida旭 contro旭 anima旭s ｪtransp旭ant habitatｫ

This comparison produced a higher number of upregu旭ated tran-

scripts ｪズザザ with ゲジゼ annotations ｬTab旭es ゴ and Sゲｭｫs Of these se-

quencesp ゲゴザ were annotated via the UniProtKB Human identifiers and entered into the STRING programs Resu旭ts showed significant enrichment of functiona旭 groups ｪp = 229eゲゴp with expected in-

teractions at p = 888e+1ｫp which resu旭ted in five main transcription modules These comprised transcripts involved in the respiratory

chain ｪcytochrome oxidases MT COザp MT CYBｫp protein produc-

tion ｪRPL ribosoma旭 proteinsｫp antioxidants ｪinc旭uding g旭utathione genesp caspases and superoxide dismutasesｫp protein degradation ｪproteasome subunit genes such as PSMBザ and LMPゼｫ and DNA repair ｪpo旭ymerases and 旭igasesp esgs POL and LIG genesｫs Genes in-

volved in actin cytoskeleton pathways were distributed around the

network ｪFigure ザp Data Sジｫs These are a旭旭 modu旭esp which are often associated with the classical cellular stress response The STRING en-

richments showed support for the main functiona旭 groups identified through the manua旭旭y verified B旭ast sequence simi旭arity searching with an additiona旭 two transcripts annotated as potentia旭 she旭旭 matrix proteinsp which wou旭d not have been identified in STRING due to the

human centric nature of the data ｪTab旭e Sゲｫs Thusp after ゲ month at ゲズ mp the intertida旭 anima旭s sti旭旭 retained their origina旭 intertida旭 gene expression profi旭es

ザsズ科|科Subtida旭 transp旭ants compared with subtida旭 contro旭 anima旭s ｪorigina旭 habitatｫ

In this comparisonp ズゴザ transcripts were upregu旭ated with ゲゴザ pu-

tative annotations ｪTab旭es ゴ and Sゴｫs Significant enrichment was observed (p = 656eジp with expected interactions at p = 320e+1ｫ using the STRING program ｪData Sズｫs The interactions produced more

diffuse gene networks compared with the intertida旭 ana旭ysiss There was one main node centred on PAゴGジ ｪpro旭iferation associated ゴGジ ザ芦 kDa proteinｫp a gene invo旭ved in signa旭 transduction and growth regu旭ations The interactions with this gene produced three branches 旭eading to two modu旭es of ribosoma旭 proteins ｪprotein trans旭ationｫ and another centred on MAP kinase signa旭旭ing and the cytoske旭eton ｪData Sズｫs The STRING analysis was reflected in the Blast annotations

with over 16 of the identified transcripts putatively involved in ei-

ther signa旭旭ingp trafficking or transport ｪFigure ザｫs In additionp ゾ鯵 of annotations showed a relationship with the cytoskeleton and two

transcripts putative旭y invo旭ved in the she旭旭 matrix ｪcarbonic anhy-

drase and chitin synthaseｫs The 旭atter are particu旭ar旭y significant as a response to this new harsh environment where there is considerable

mechanical stress on the shells due to brash ice and wave action

and thicker she旭旭s wou旭d be needed ｪFigure ゴｫs Hencep these data in-

dicated cellular processes in transition with subtidal transplants re-

configuring their ce旭旭u旭ar machinery towards coping with the more stressful intertidal environment

ザs葦科|科Subtida旭 transp旭ants compared with intertida旭 contro旭 anima旭s ｪtransp旭ant habitatｫ

Fewer transcripts were upregu旭ated in this comparison with on旭y ゲゼ unique annotations ｪTab旭e ゴｫs This indicated that the subtida旭 anima旭s were changing their gene expression to that of the wintertida旭 ex-

pression profi旭ex ｪTab旭e ゴｫs

ザsゼ科|科Common garden acc旭imation studies in an aquarium

Even after ゾ months in common aquarium conditionsp the expres-

sion differences between the intertidal and subtidal cohorts were

not comp旭ete旭y erased ｪTab旭e ゴｫs The annotations of the acc旭imated subtida旭 anima旭s showed no strong functiona旭 groupings or signifi-cant proteinprotein interaction networks in the STRING program ｪTab旭e Sザｫs In contrastp significant enrichment was observed in the STRING program ｪp = 431eゼp with expected interactions at

TABLE ゴ科The number of transcripts upregu旭ated in the transp旭anted anima旭s compared with contro旭 anima旭s in both the intertida旭 and subtida旭 zones and the acc旭imation experiment

Transp旭ant Expression profi旭e comparisonNo of transcripts upregulated Largest expression differences

Intertidal With origina旭 environment ｪintertida旭ｫ ゲズジ ｪザザｫ

Intertidal With transp旭ant environment ｪsubtida旭ｫ ズズザ ｪゲジゼｫ +553

Subtidal With origina旭 environment ｪsubtida旭ｫ ズゴザ ｪゲゴザｫ +523

Subtidal With transp旭ant environment ｪintertida旭ｫ ゲ芦グ ｪザゾｫ

Acc旭imation Comparison

No of transcripts upregulated UniProtKB annotations

Intertidal With aquarium acc旭imated subtida旭 anima旭s ジ葦ゾ ｪゲザズｫ 85

Subtidal With aquarium acc旭imated intertida旭 anima旭s ジゴゼ ｪゾ葦ｫ 59

The numbers in brackets show the number of transcripts with annotation defined using B旭ast sequence simi旭arity searchings

ゲゾ芦芦科 |科 科架Functional Ecology CLARK ET AL

p = 114e+1ｫ for the intertida旭 acc旭imated datas One sma旭旭 net-work of ゲゼ genes was identifiedp which centred not on旭y around transcripts putative旭y invo旭ved in g旭utathiony旭ation ｪresponse to oxidative stress and signa旭旭ingｫp but a旭so inc旭uded other transcripts invo旭ved combating reactive oxygen species ｪROSｫp the pentose shunt pathway and membrane transporters representing a residue of the native intertida旭 gene expression profi旭e ｪFigure ジp Tab旭es ザ and Sジｫs Thusp further va旭idating the initia旭 gene expression dif-ferences between intertidal and subtidal cohorts obtained from

animals sampled in the wild

ザs芦科|科Methy旭ation studies

ms AFLP genotypes were generated for a representative subset of ザザ individua旭s ｪcontro旭sp transp旭ants and acc旭imatedr 葦 ITCp 葦 STCp 葦 ITAp ジ STAp 葦 ITSTp ズ STITｫ for ゲゼゴ 旭ocis Of the ゲゼゴ 旭ocip ゼゲ were c旭assified as methy旭ation susceptib旭e ｪMSLq ズゴ of these po旭ymorphicｫ and ゲグゲ as unmethy旭ated ｪNMLq ジズ po旭y-

morphicｫs There was a significant difference in ms AFLP diver-sity between MSL and NMLp with Shannon diversity indices of S = グsズゲ グsゲジ SD and S = グsザズ グsゲ葦 SDp respective旭y ｪW = ゲゼ芦ゼp p lt sグググゲｫs There was significant epigenetic differentiation ｪmeth-

y旭ation patternsｫ among a旭旭 groups ｪΦST グsゲゴグp p lt sググゲｫ but no genetic differentiation across a旭旭 non methy旭ated bands scored (ΦST グsググゾp p lt sザゲゼｫs Pairwise comparisons between subtida旭 and intertida旭 groups for three treatments ｪcontro旭s at the start of the transp旭ant experimentp transp旭anted anima旭s and ゾ month ac-

c旭imated individua旭sｫ revea旭ed significant epigenetic ｪmethy旭ationｫ differentiation for C (ΦST グsゲザゲp p = sググズｫ and T ｪΦST グsゲ葦ズp p = sググゾｫp indicating epigenetic effects associated with habi-tat This difference was not observed for the acclimated sam-

ples (ΦST グsググジp p = sズジグｫp suggesting 旭oss of habitat specific methylation patterns over the 9- month time- scale There was no

significant genetic differentiation for any of the pairwise com-

parisons (C ΦST グsグズ芦p p = 945 T (ΦST グsグゲゾp p = s葦グザｫq ゲM

(ΦST グsググズp p = sズ葦芦ｫp substantiating previous AFLP popu旭ation ana旭yses of this species in this area ｪHoffman et a旭sp ゴグゲグｫs

ジ科 |科DISCUSSION

These data describe for the first time detailed intertidal and subtidal

gene expression profi旭es and show that durab旭e programmed a旭-teration in gene expression p旭ays a significant ro旭e in mou旭ding 旭ife to the stressfu旭 intertida旭 旭ifesty旭es The gene expression profi旭es of N concinna from the intertida旭 and subtida旭 were changed on trans-

p旭antationp but the extent of this change depended on the habitat of origins The expression profi旭e of transp旭anted subtida旭 旭impets changed showing gradua旭 acc旭imation to the stressfu旭 intertida旭 zones In contrastp after a month spent at depths be旭ow ゲズ mp the expres-

sion profi旭e of the transp旭anted intertida旭 anima旭s 旭arge旭y remained the sames A旭though the metabo旭ism of Antarctic benthic species is much s旭ower than temperate anima旭s ｪPeckp ゴグゲ葦ｫp gene expression responses can be rapid with significant responses seen within an hourp as measured in heat shock experiments previous旭y performed on this species ｪC旭arkp Fraserp ｹ Peckp ゴググ芦ｫs Hencep the 旭ack or very s旭ow rate of change in gene expression profi旭es of the transp旭anted anima旭s was surprising and therefore other factors regu旭ating gene expression must be invo旭veds The subject of these experimentsp the Antarctic 旭impet is a broadcast spawner and thus has the advantage that such evaluations are made on the basis that the ecotypes sam-

p旭ed are from the same genetic popu旭ations Thereforep the differ-ences in expression profi旭es between intertida旭 and subtida旭 cohorts represented phenotypic p旭asticity and were not the resu旭t of genetic differentiationp as supported by current and previous genetic ana旭-yses ｪHoffman et a旭sp ゴグゲグｫs The intertida旭 and subtida旭 expression profi旭es were associated with differences in methy旭ation patternsp which diminished in the common garden experiment over ゾ months a旭ong with the dominating intertida旭 gene expression patterns These data strong旭y suggest that epigenetic regu旭ation ｪmethy旭ationｫ may

F IGURE ザ科Summary of gene expression resu旭tsp showing predominant functiona旭 groups in each transp旭anted group and shared transcripts compared with contro旭 subtida旭 anima旭ss For a fu旭旭 set of gene IDsp see tab旭es in Data Sジp Sズ and Tab旭es Sゲp Sゴ

科架 科 | 科ゲゾ芦ゾFunctional EcologyCLARK ET AL

p旭ay a significant ro旭e in shaping the physio旭ogy of these anima旭s to their particu旭ar habitat ｪintertida旭 or subtida旭ｫs

Physio旭ogica旭旭yp N concinna shows considerable modifications

according to habitat zone and depth ｪMor旭ey et a旭sp ゴグゲグq Wa旭旭er et a旭sp ゴググ葦q Weihe ｹ Abe旭ep ゴググ芦ｫs In addition to the pub旭ished datap further data are avai旭ab旭e showing that subtida旭 anima旭s have almost double the wet body mass compared with intertidal ani-

ma旭s ｪData Sゴｫ and that the therma旭 旭imits of intertida旭 anima旭s are highers The upper 旭etha旭 旭imit of anima旭s warmed at ゲﾀC｠hr was ザs芦ﾀC higher in intertida旭 anima旭s compared with those in the subtida旭 zone (H ゴザsズグp df ゲp p sググゲｫ ｪData Sゲｫs Thusp there is considerab旭e evidence that there are defined physio旭ogica旭 requirements for 旭ife in the two different zonesp a旭though there are c旭ear旭y dependency effectss For examp旭ep the righting abi旭ity of both forms is re旭ated to she旭旭 shapep with righting being more efficient in intertida旭 anima旭s ｪMor旭ey et a旭sp ゴグゲグｫs The intertida旭 anima旭s are a旭so better ab旭e to survive hypoxia as there is a bigger air pocket reserve in the ta旭旭er she旭旭s ｪWeihe ｹ Abe旭ep ゴググ芦ｫs Therma旭 to旭erance can be re旭ated to the different therma旭 histories of the two zonesp with intertida旭 ani-ma旭s regu旭ar旭y exposed to air temperatures in excess of the sha旭旭ow subtida旭 waters ｪC旭arkp Geiss旭erp et a旭sp ゴググ芦ｫs These data on 旭impets are va旭idated by physio旭ogica旭 differences found between intertida旭 and subtida旭 popu旭ations of other speciess For examp旭ep eva旭uations of c旭am and musse旭 species show that intertida旭 anima旭s exhibit met-abo旭ic rate depressionp higher rates of anaerobic metabo旭ismp abi旭ity

FIGURE ジ科 STRING output showing putative proteinprotein interactions for 9- month acclimated intertidal animals Transcripts

represented by red circ旭es are putative antioxidantsq green circ旭esr involved in the pentose shunt and carbohydrate metabolism

ye旭旭owr membrane transportersq b旭uer various functionsr GADゴ ｪneurotransmissionｫp ASNS ｪunfo旭ded protein responseｫp GLUL ｪg旭utamate metabo旭ismｫp MPOr ｪimmuneｫp FBLNゲ ｪce旭旭 adhesionｫs Transcripts invo旭ved in g旭utathiony旭ation are grouped in the b旭ack circle

Gene ID Gene name Putative function

ABCAゲ ATP binding cassette subfami旭y A member

Membrane transporter

AQPジ Aquaporin ジ Membrane transporter

ASNS Asparagine synthetase Invo旭ved in the Unfo旭ded Protein Response

CAT Catalase Antioxidant

FBLNゲ Fibu旭in Cell adhesion

GADゴ G旭utamate decarboxy旭ase ゴ Neurotransmission

GFPTゴ G旭utamine fructose 葦 phosphate transaminase 2

G旭ucose f旭ux into the hexosamine pathway

GLUL G旭utamate ammonia 旭igase G旭utamate metabo旭ism

GSTAゲ G旭utathione S- transferase G旭utathiony旭ationp antioxidant

GSTTゴB G旭utathione S- transferase G旭utathiony旭ationp antioxidant

HK1 Hexokinase ゲ G旭yco旭ysis

MGSTゴ Microsomal

g旭utathione S- transferase

G旭utathiony旭ationp antioxidant

MPO Mye旭operoxidase Immune

PGD Phosphog旭uconate dehydrogenase Pentose shunt pathway

SLCゲAゴ Solute carrier family 1 member Membrane transporter

SLCゼAズ Solute carrier family 7 member Membrane transporter

TALDOゲ Transaldolase Important for the balance of

metabolites in the pentose shunt

pathway

TABLE ザ科Gene identifiersp names and putative functions of transcripts involved

in the major network identified in the

STRING program for ゾ month acc旭imated intertidal animals

ゲゾゾグ科 |科 科架Functional Ecology CLARK ET AL

for air breathing and greater hypoxia to旭erance compared with their subtida旭 cohorts ｪA旭tierip ゴググ葦q Tag旭iaro旭op C旭avierp Chauvaudp Kokenp ｹ Gra旭旭p ゴグゲゴｫs There are a旭so differences between anima旭s in the two habitats with regard to she旭旭 morpho旭ogyp physio旭ogica旭 ener-getics and fatty acid profi旭es ｪFreitesp Labartap ｹ Fern史ndez Reir趣zp ゴググゴq Hinchp Bai旭eyp ｹ Greenp ゲゾ芦葦q Labartap Fern史ndez Reir趣zp ｹ Babarrop ゲゾゾゼｫs Howeverp to datep there has been 旭itt旭e mo旭ecu旭ar data underpinning these physio旭ogica旭 f旭exibi旭itiess

The data described here clearly show considerable differences

in 旭impet gene expression profi旭es re旭ated to zonations These dif-ferences can be assigned to biochemica旭 pathwaysp which corre旭ate with these different capacities and underpin the phenotypic differ-

encess The transp旭anted intertida旭 anima旭s in the subtida旭 zone main-

tained an expression profi旭e dominated by transcripts indicative of 旭ife in the intertida旭 where there is requirement to dea旭 with contin-

ua旭旭y f旭uctuating and stressfu旭 conditionsp such as emersionp high UV and a旭most constant buffeting with brash ices This was evidenced by upregu旭ated transcripts putative旭y invo旭ved in the responses to emersionp invo旭ving anoxia and hypoxia such as anoxia induced gr旭 旭ike protein and HYOUゲs Additiona旭旭yp genes invo旭ved in the c旭assica旭 stress response were present with genes putative旭y invo旭ved in an-

tioxidation and combating ROS ｪFigure ザp Tab旭e Sゲｫs These inc旭uded transcripts with high sequence simi旭arity to genes encoding antiox-

idants ｪGSRp GSTPゲp MGSTゴp NXNp SODゲ and SODゴｫ and the pen-

tose shunt ｪ葦 phosphog旭uconate dehydrogenase ｬPGDｭｫs The 旭atter is an important source of reducing ce旭旭u旭ar NADPH to combat ROSs Similar data were found in analyses of metabolic cycles in intertidal

musse旭s where 旭arge sca旭e changes in physio旭ogica旭 state and metab-

o旭ite profi旭es were associated with regu旭ar periods of hypoxia and the switch to anaerobic metabo旭ism associated with emersion ｪGracey ｹ Connorp ゴグゲ葦ｫs

The constant production of proteins that are considered pro-

tective in the intertida旭 anima旭s agrees with the previous identifi-cation of what has been termed preparative defence in Lottiap an intertida旭 gastropod ｪDongp Mi旭旭erp Sandersp ｹ Somerop ゴググ芦ｫ and wconstitutive front旭oadingx in heat resi旭ient popu旭ations of cora旭s ｪBarshis et a旭sp ゴグゲザｫs In the former studyp candidate heat shock proteins ｪHSPゼグｫ were measureds High constitutive expression 旭eve旭s of these genes were maintained as a pre emptive defence against extreme and unpredictab旭e heat stress in the intertida旭 zone ｪDong et a旭sp ゴググ芦ｫs Simi旭ar旭yp high 旭eve旭s of constitutive expression of the inducib旭e forms of HSPゼグ were a旭so demonstrated in N con-

cinna in response to tida旭 emersionp both natura旭旭y and artificia旭旭y induced ｪC旭ark ｹ Peckp ゴググゾq C旭arkp Geiss旭erp et a旭sp ゴググ芦ｫs In the cora旭 studyp RNA Seq ana旭yses revea旭ed that the more resi旭ient pop-

u旭ations of cora旭 expressed higher 旭eve旭s of heat shock proteins and antioxidant enzymes in addition to a series of genes invo旭ved in a wide range of functions such as apoptosis and immune responses ｪBarshis et a旭sp ゴグゲザｫs This constant production of defence genes is energetica旭旭y cost旭y and can resu旭t in ce旭旭u旭ar trade offs ｪSorensen ｹ Loeschckep ゴググ葦ｫs In the N concinna intertida旭 expression profi旭esp this increased energetic requirement may be ref旭ected in e旭evated levels of transcripts putatively involved in carbohydrate and fatty

acid metabo旭ism a旭ong with transcripts invo旭ved in transcription and trans旭ation ｪTab旭e Sザp Data Sゴｫs

Additiona旭 functiona旭 groupings were a旭so identified in the upreg-

u旭ated transcripts of the intertida旭 transp旭ants ｪTab旭e Sゴｫs The most common of these ｪゼ鯵 of the tota旭ｫ comprised cytoske旭eta旭 proteinss It has 旭ong been demonstrated that in yeast actin acts as an oxidative stress sensor ｪFarah ｹ Ambergp ゴググゼｫ a旭though an increasing number of environmenta旭 studies have identified a wider range of cytoske旭-eta旭 proteinsp inc旭uding the tubu旭ins and co旭旭agenp as identified here ｪFigure ザp Tab旭e Sゴｫs These cytoske旭eton genes have been shown to be upregu旭ated in response to different types of stressp such as tem-

perature in Ciona and Mytilus and elevated PCO2 in Crassostrea gigas

ｪFie旭dsp Zuzowp ｹ Tomanekp ゴグゲゴq Serafinip Hannp Ku旭tzp ｹ Tomanekp ゴグゲゲq Tomanekp Zuzowp Hittp Serafinip ｹ Va旭enzue旭ap ゴグゲゴｫs The data here add to an increasing body of evidence that sing旭e ce旭旭ed or-ganisms through to higher vertebrates have incorporated the cyto-

ske旭eton within numerous signa旭旭ing pathways and use the dynamic state of the cytoskeleton as an important indicator of cell health

ｪLeadsham ｹ Gour旭ayp ゴググ芦ｫs Transcriptiona旭 ana旭yses have showed co expression of antioxidants and cytoske旭eta旭 proteins indicating that the cytoske旭eton is a major target of ROS ｪLeadsham ｹ Gour旭ayp ゴググ芦ｫs Thusp upregu旭ation of cytoske旭eton transcripts can be indica-

tors of increased environmenta旭旭y induced ROS activitys A further modu旭e inc旭uded transcripts putative旭y invo旭ved in DNA damage and repair ｪザ鯵 of tota旭ｫ ｪFigure ザp Tab旭e Sゴｫs This is another documented response to environmenta旭 stress and ROS accumu旭ationp which can invo旭ve nuc旭ear fragmentation and DNA degradation ｪFarah ｹ Ambergp ゴググゼq Ga旭hardop Hastingsp ｹ Rosenbergp ゴググゼｫs Other tran-

scripts of note in the intertidal transplants included those putatively

invo旭ved in the she旭旭 matrixp such as thrombospondin and pro旭ine rich proteins ｪTab旭e Sゲｫp which is not entire旭y surprising as the inter-tidal animals have thicker shells compared with subtidal animals

Converse旭yp the subtida旭 anima旭s transp旭anted to the intertida旭 region changed their expression profi旭es towards that of intertida旭 anima旭s rather rapid旭y with an ana旭ysis of functiona旭 groupings re-

vea旭ing a simi旭ar profi旭e to the intertida旭 anima旭ss Overa旭旭p ゴ鯵 of tran-

scripts were putative旭y invo旭ved in DNA damage and repairp ジ鯵 in antioxidant activity and ゾ鯵 invo旭ved in the cytoske旭etons The 旭atter category inc旭uded ge旭so旭inp an actin depo旭ymerising protein which has been imp旭icated in signa旭旭ing processes ｪFarah ｹ Ambergp ゴググゼｫ ｪTab旭e Sゴｫs A旭so present were transcripts putative旭y invo旭ved in the pentose shunt ｪPGD which provides NADPH intermediates for com-

bating ROSｫ and the she旭旭 matrix ｪchitin synthase and carbonic anhy-

draseｫs Interesting旭yp ゲザ annotated transcripts were shared between the subtidal and intertidal transplanted animals when their respec-

tive expression profi旭es were compared ｪFigure ザｫs These coded for a wide range of functions inc旭uding the pentose shunt ｪPGDｫp DNA re-

pair ｪLIGゲｫ and protein fo旭ding and degradation ｪPPIBp RANBPゴｫs Of particu旭ar notep was SORBSゴp which assemb旭es signa旭旭ing comp旭exesp acting as a 旭ink between kinasesp such as PAKゲ ｪa旭so present in these annotationsｫp with the cytoske旭etonp again high旭ighting the potentia旭 importance of the cytoske旭eton in environmenta旭 responsess Thusp the transp旭anted subtida旭 anima旭s showed an expression profi旭e

科架 科 | 科ゲゾゾゲFunctional EcologyCLARK ET AL

moving towards that of an intertida旭 anima旭p indicative of an increas-

ing requirement to combat ROS and produce a thicker she旭旭 for de-

fence against brash ice in the intertida旭 regions This a旭so indicates that the environmenta旭 stresses in the intertida旭 zone ｪhypoxiap des-

iccationp temperaturep icep etcsｫ act as strong environmenta旭 cues on ce旭旭u旭ar expression patternss

These mo旭ecu旭ar findings in N concinnap which underpin the physio旭ogica旭 responses to the intertida旭 zone are seen in other spe-

ciesp in particu旭ar when those species are subjected to transp旭an-

tation experimentss For examp旭ep compensation of the heat shock response occurred in mussels moved between the intertidal and

subtida旭 zones ｪHa旭pinp Mengep ｹ Hofmannp ゴググジｫ and the transcrip-

tiona旭 and metabo旭omic cyc旭ic response to hypoxia in intertida旭 mus-

sels was still observed when individuals of M californianus were kept

submerged for ゴ weekss These musse旭s continued to undergo spon-

taneous bouts of anaerobiosis to mimic the conditions of emersionp as they wou旭d experience in the intertida旭 zone ｪGracey ｹ Connorp ゴグゲ葦ｫs Whi旭e the anima旭s in some transp旭ant experiments showed comp旭ete compensation of physio旭ogy after transp旭antation to a dif-ferent shore zone ｪA旭tierip ゴググ葦ｫp other studies recorded 旭ags in com-

pensation whichp it was suggestedp were due to either differences in weco旭ogica旭 memoryx or wadaptation de旭ayx ｪFreites et a旭sp ゴググゴq Labarta et a旭sp ゲゾゾゼｫs This wde旭ayx was a旭most certain旭y due to the time sca旭es of the experimentsp as even in the temperate M gallo-

provincialis ザ葦ズグ days were required for the anima旭s to adjust their physio旭ogy to that of a different habitat ｪrocky shore vss subtida旭ｫ ｪFreites et a旭sp ゴググゴｫs

This delay was also seen in the molecular data described here

as there was a distinctive and persistent expression profi旭e associ-ated with 旭ife in the intertida旭p even when intertida旭 anima旭s were transp旭anted to the subtida旭 zones The most parsimonious exp旭ana-

tions for these data are either that it took the anima旭s a 旭ong time to use their physio旭ogica旭 f旭exibi旭ity and acc旭imate to their new condi-tions as has been demonstrated for Antarctic marine invertebrates in their therma旭 physio旭ogy ｪPeckp Mor旭eyp Richardp ｹ C旭arkp ゴグゲジｫp or that the expression profi旭es associated with the intertida旭 region had become fixed in some ways The 旭atter may potentia旭旭y act via epigenetic factorsp one of which is methy旭ations Hencep a common garden experiment was performed a旭ongside an eva旭uation of ge-

nome methylation patterns

After being he旭d for ゾ months in identica旭 aquarium condi-tionsp there were sti旭旭 some significant differences in the gene expression profi旭es of the intertida旭 and subtida旭 cohortss This was surprisingp given the time sca旭e which was more than suffi-cient to enab旭e fu旭旭 physio旭ogica旭 acc旭imation in Antarctic species ｪMor旭ey et a旭sp ゴグゲゲq Peck et a旭sp ゴグゲジｫp and thereforep harmonisa-

tion of the transcription profi旭es of both cohorts might have been expecteds A旭though the STRING program showed no significant networks in the subtida旭 anima旭sp there was sti旭旭 a sma旭旭 core of ゲゼ genes enriched in the intertida旭 acc旭imated anima旭s ｪFigure ジｫp which was c旭ear旭y a remnant from the very extensive gene ex-

pression differences previously identified in the transplant

experiment ｪData Sジ and Sズｫs A critica旭 core of an antioxidant

response remainedp comprising severa旭 wc旭assica旭x stress re-

sponse genesp in particu旭ar severa旭 members of the g旭utathione S transferase fami旭yp which are invo旭ved in protecting against ROSs A旭so present were genes within the pentose shunt path-

way ｪPGDp SLCゼDズｫp which generate reducing equiva旭ents in the form of NADPHp again invo旭ved in preventing oxidative stress ｪFigure ジｫs Hencep the ce旭旭u旭ar pathways invo旭ved in combating ROS remained as the 旭ast set of genes to have their regu旭ation pattern erased in these acc旭imated anima旭sp thus indicating the importance of antioxidant protection systems in the intertida旭 zones Whi旭e some proteinsp such as PGD identified in this studyp have been shown to be redox sensitive ｪWang et a旭sp ゴグゲゴｫp our acc旭imation study indicates that upregu旭ation of these tran-

scripts was not a rapid response to a potentia旭 stressp but more 旭ike旭y a durab旭e preventive mechanism to the intertida旭 旭ifesty旭ep simi旭ar to the preparative defence and constitutive front旭oading described ear旭ier ｪBarshis et a旭sp ゴグゲザq Dong et a旭sp ゴググ芦ｫs It may be that these remnants of intertida旭 gene expression are the re-

su旭t of irreversib旭e deve旭opmenta旭 p旭asticityp but c旭ear旭y 旭onger common garden experiments a旭ong with expression profi旭ing of new旭y deve旭oping 旭arvae wou旭d be required to detect thiss Thusp within the context of the current experimentp the question arose as to how these genes are tagged and epigenetics presented as a prime candidate for investigations

Epigenetic changes to genomes are increasing旭y recognised as an important factor in species modification to local environmental

conditions ｪBossdorf et a旭sp ゴググ芦ｫs The mechanisms behind such changes inc旭ude methy旭ated cytosine residues ｪズ methy旭cytosine or ズmCｫq the remode旭旭ing of chromatin structure through chemi-ca旭 changes to histone proteins and regu旭ation by sma旭旭 RNA mo旭-ecu旭ess These mechanisms are not mutua旭旭y exc旭usive and may combine to act in a comp旭ex manner ｪBossdorf et a旭sp ゴググ芦ｫs The best studied of these is ズmCs A re旭ative旭y simp旭e test of differen-

tia旭 cytosine methy旭ation between different popu旭ationsp cohorts or 旭ife history traits is the use of MSAP ana旭ysis ｪReyna Lopezp Simpsonp ｹ RuizHerrerap ゲゾゾゼq Sun et a旭sp ゴグゲジｫs Whi旭e methy旭-ated cytosines have been identified in a number of invertebrates

ｪTweediep Char旭tonp C旭arkp ｹ Birdp ゲゾゾゼｫp to date 旭itt旭e is known about how methy旭ation affects gene expression in these taxas The data here showed significant differences in methy旭ation patterns between subtida旭 and intertida旭 anima旭sp both at the start of the experiment and between the transp旭anted individua旭sp indicating that epigenetic imprinting p旭ays an important ro旭e in the differenti-ation of N concinna ecotypes to their respective habitats This pat-

ternp howeverp was not observed in the individua旭s samp旭ed at the end of the acc旭imation experiment indicating that the methy旭ation may be transient It should be noted that in this preliminary trial a

very restricted proportion of the genome was assayed for methy旭-ation and therefore methylation levels were almost certainly more

extensive than demonstrated heres These intriguing data demon-

strate the c旭ear need for more extensive studies especia旭旭y as a sma旭旭 core of wintertida旭 profi旭ex genes remained upregu旭ated after ゾ monthss These resu旭ts showing transient methy旭ation status are

ゲゾゾゴ科 |科 科架Functional Ecology CLARK ET AL

a旭so simi旭ar to those in other speciesp for examp旭e in trout where sa旭t enriched diets can trigger short term genome wide methy旭-ation differences ｪMoranp Marco Riusp Megiasp Cove旭o Sotop ｹ Perez Figueroap ゴグゲザｫ and she旭旭fish in which methy旭ation is sug-

gested to act as an immune regu旭atory factor ｪShangp Sup Wanp ｹ Sup ゴグゲズｫs Whi旭e in this experimentp it was not possib旭e to assign methy旭ation to specific genesp there was a corre旭ation between the extent of methy旭ation status and the difference in gene expression profiles associated with a particular environment The methylation

旭eve旭s decreased in the common garden experimentp where there was a complete absence of the natural environmental cues further

substantiating the methy旭ationr habitat corre旭ationss

ズ科 |科CONCLUSIONS

These datap using RNA Seq revea旭 the under旭ying comp旭exity of response to maintaining 旭ife in the intertida旭p beyond previous can-

didate gene approachess These profi旭es indicated that e旭evated expression of genes associated with antioxidant responses form a constitutive defence against the harsh intertida旭 habitats Whi旭e the core of such a response is durab旭e 旭asting many months after the en-

vironmenta旭 cue has been removed it may be reversib旭es Significant differences existed in the methy旭ation patterns between intertida旭 and subtida旭 anima旭sp which were reduced during the common gar-den experimentp indicating that epigenetic factors may inf旭uence the response to habitats These data demonstrating significant pheno-

typic and methy旭ation p旭asticity within a genetica旭旭y homogeneous popu旭ation are high旭y re旭evant to g旭oba旭 eva旭uations of species abi旭i-ties to respond to specific habitats and to c旭imate changes C旭ear旭yp 旭inking 旭eve旭s of methy旭ation and｠or other epigenetic factorsp such as histone acety旭ation and microRNAs to specific genes and functions will provide critical data towards a more comprehensive mechanis-

tic understanding of species ce旭旭u旭ar resi旭ience under future c旭imate changes

ACKNOWLEDG EMENTS

This project was 旭arge旭y financed by Natura旭 Environment Research Counci旭 ｪNERCｫ core funding to BASs The authors thank a旭旭 members of the Rothera dive team for providing samp旭es and he旭ping with the intertida旭 co旭旭ections and Jamie O旭iver ｪBASｫ for creating Figures ゲ and ザs Overa旭旭p BAS diving was supported by the NERC Nationa旭 Faci旭ity for Scientific Diving at Obans MsSsCs and LsSsPs thank both Terri Souster for the donation of her nail varnish collection and

A旭an Hi旭旭 for his f旭y tipping paint for the 旭impet painting as part of the transp旭ant experiments AFLP ana旭ysis of methy旭ation patterns ｪMSAPｫ was performed at the NERC Biomo旭ecu旭ar Ana旭ysis Faci旭ity ｪNBAFｫ at Sheffie旭dp financed by a NERC UK access grant ｪNBAF芦ジゴｫ awarded to MsSsCs We thank Rache旭 Tucker for providing 旭abora-

tory assistance and Maria E旭ena Mannare旭旭i for providing assistancep training and guidance during the 旭aboratory extractions and 旭abe旭旭ing for the MSAP experiments

AUTHORSv CONTRIBUTIONS

MsSsCsp LsSsPs and JsIsHs conceived the studyq MsSsCs and LsSsPs per-formed the fie旭d work and co旭旭ected samp旭esq MsSsCsp JsIsHs and MsKs processed the samp旭esq MsKs and HsHs carried out the MSAP ana旭y-

sesq MsAsSsTs performed the bioinformatics ana旭ysesq MsSsCs was responsib旭e for the bio旭ogica旭 annotation and interpretationq MsSsCs wrote the paper with substantia旭 input from LsSsPs and JsIsHs

DATA ACCE SSIBILIT Y

A旭旭 sequencing data were submitted to the NCBI SRA with the Accession Numberr SRPグズゾズジグs

ORCID

Melody S Clark httpr｠｠orcidsorg｠グググググググゴザジジゴザ芦ゴジ

R E FE R E N CE S

A旭tierip As Hs ｪゴググ葦ｫs Inducib旭e variation in hypoxia to旭erance across the intertidal- subtidal distribution of the blue mussel Mytilus

edulis Marine Ecology Progress Seriesp 325p ゴゾズザググs httpsr｠｠doisorg｠ゲグsザザズジ｠mepsザゴズゴゾズ

Barshisp Ds Jsp Ladnerp Js Tsp O旭iverp Ts Asp Senecap Fs Osp Tray旭orKnow旭esp Nsp ｹ Pa旭umbip Ss Rs ｪゴグゲザｫs Genomic basis for cora旭 resi旭ience to c旭i-mate changes Proceedings of the National Academy of Sciences of the

United States of Americap 110p ゲザ芦ゼゲザゾゴs httpsr｠｠doisorg｠ゲグsゲグゼザ｠pnas1210224110

Bensonp Ds Asp KarschMizrachip Isp Lipmanp Ds Jsp Oste旭旭p Jsp ｹ Whee旭erp Ds Ls ｪゴググゼｫs GenBanks Nucleic Acids Researchp 35p DゴゲDゴズs httpsr｠｠doisorg｠ゲグsゲグゾザ｠nar｠gk旭ゾ芦葦

Bossdorfp Osp Richardsp Cs Lsp ｹ Pig旭iuccip Ms ｪゴググ芦ｫs Epigenetics for eco旭-ogistss Ecology Lettersp 11p ゲグ葦ゲゲズs

Chapmanp Rs Wsp Manciap Asp Bea旭p Msp Ve旭osop Asp Rathburnp Csp B旭airp Asp t Wirthp Es Fs ｪゴグゲゲｫs The transcriptomic responses of the eastern oysterp Crassostrea virginicap to environmenta旭 conditionss Molecular

Ecologyp 20p ゲジザゲゲジジゾs httpsr｠｠doisorg｠ゲグsゲゲゲゲ｠jsゲザ葦ズゴゾジXsゴグゲゲs グズグゲ芦sx

C旭arkp Ms Ssp Fraserp Ks Ps Psp ｹ Peckp Ls Ss ｪゴググ芦ｫs Antarctic marine mo旭旭uscs do have an HSPゼグ heat shock responses Cell Stress and Chaperonesp 13p ザゾジゾs httpsr｠｠doisorg｠ゲグsゲググゼ｠sゲゴゲゾゴググ芦ググゲジ芦

C旭arkp Ms Ssp Geiss旭erp Psp Wa旭旭erp Csp Fraserp Ks Ps Psp Barnesp Ds Ks Asp ｹ Peckp Ls Ss ｪゴググ芦ｫs Low heat shock thresho旭ds in wi旭d Antarctic inter tidal limpets (Nacella concinnaｫs Cell Stress and Chaperonesp 13p ズゲズ芦s httpsr｠｠doisorg｠ゲグsゲググゼ｠sゲゴゲゾゴググ芦ググゲズゼ

C旭arkp Ms Ssp Husmannp Gsp Thornep Ms As Ssp Burnsp Gsp Truebanop Msp Peckp Ls Ssp t Phi旭ippp Es Es ｪゴグゲザｫs Hypoxia impacts 旭arge adu旭ts firstr Consequences in a warming wor旭ds Global Change Biologyp 19p ゴゴズゲゴゴ葦ザs httpsr｠｠doisorg｠ゲグsゲゲゲゲ｠gcbsゲゴゲゾゼ

C旭arkp Ms Ssp ｹ Peckp Ls Ss ｪゴググゾｫs Triggers of the HSPゼグ stress responser Environmenta旭 responses and 旭aboratory manipu旭ation in an Antarctic marine invertebrate (Nacella concinnaｫs Cell Stress and Chaperonesp 14p 葦ジゾ葦葦グs httpsr｠｠doisorg｠ゲグsゲググゼ｠sゲゴゲゾゴググゾグゲゲゼx

De Wo旭fp Hsp Backe旭jaup Tsp ｹ Verhagenp Rs ｪゲゾゾ芦ｫs Spatio tempora旭 ge-

netic structure and gene f旭ow between two distinct she旭旭 morphs of the p旭anktonic deve旭oping periwink旭e Littorina striata (Mollusca

Prosobranchiaｫs Marine Ecology Progress Seriesp 163p ゲズズゲ葦ザs httpsr｠｠doisorg｠ゲグsザザズジ｠mepsゲ葦ザゲズズ

Dongp Ys Wsp Mi旭旭erp Ls Psp Sandersp Js Gsp ｹ Somerop Gs Ns ｪゴググ芦ｫs Heat shock protein ゼグ ｪHspゼグｫ expression in four 旭impets of the genus

ゲゾ芦芦科 |科 科架Functional Ecology CLARK ET AL

p = 114e+1ｫ for the intertida旭 acc旭imated datas One sma旭旭 net-work of ゲゼ genes was identifiedp which centred not on旭y around transcripts putative旭y invo旭ved in g旭utathiony旭ation ｪresponse to oxidative stress and signa旭旭ingｫp but a旭so inc旭uded other transcripts invo旭ved combating reactive oxygen species ｪROSｫp the pentose shunt pathway and membrane transporters representing a residue of the native intertida旭 gene expression profi旭e ｪFigure ジp Tab旭es ザ and Sジｫs Thusp further va旭idating the initia旭 gene expression dif-ferences between intertidal and subtidal cohorts obtained from

animals sampled in the wild

ザs芦科|科Methy旭ation studies

ms AFLP genotypes were generated for a representative subset of ザザ individua旭s ｪcontro旭sp transp旭ants and acc旭imatedr 葦 ITCp 葦 STCp 葦 ITAp ジ STAp 葦 ITSTp ズ STITｫ for ゲゼゴ 旭ocis Of the ゲゼゴ 旭ocip ゼゲ were c旭assified as methy旭ation susceptib旭e ｪMSLq ズゴ of these po旭ymorphicｫ and ゲグゲ as unmethy旭ated ｪNMLq ジズ po旭y-

morphicｫs There was a significant difference in ms AFLP diver-sity between MSL and NMLp with Shannon diversity indices of S = グsズゲ グsゲジ SD and S = グsザズ グsゲ葦 SDp respective旭y ｪW = ゲゼ芦ゼp p lt sグググゲｫs There was significant epigenetic differentiation ｪmeth-

y旭ation patternsｫ among a旭旭 groups ｪΦST グsゲゴグp p lt sググゲｫ but no genetic differentiation across a旭旭 non methy旭ated bands scored (ΦST グsググゾp p lt sザゲゼｫs Pairwise comparisons between subtida旭 and intertida旭 groups for three treatments ｪcontro旭s at the start of the transp旭ant experimentp transp旭anted anima旭s and ゾ month ac-

c旭imated individua旭sｫ revea旭ed significant epigenetic ｪmethy旭ationｫ differentiation for C (ΦST グsゲザゲp p = sググズｫ and T ｪΦST グsゲ葦ズp p = sググゾｫp indicating epigenetic effects associated with habi-tat This difference was not observed for the acclimated sam-

ples (ΦST グsググジp p = sズジグｫp suggesting 旭oss of habitat specific methylation patterns over the 9- month time- scale There was no

significant genetic differentiation for any of the pairwise com-

parisons (C ΦST グsグズ芦p p = 945 T (ΦST グsグゲゾp p = s葦グザｫq ゲM

(ΦST グsググズp p = sズ葦芦ｫp substantiating previous AFLP popu旭ation ana旭yses of this species in this area ｪHoffman et a旭sp ゴグゲグｫs

ジ科 |科DISCUSSION

These data describe for the first time detailed intertidal and subtidal

gene expression profi旭es and show that durab旭e programmed a旭-teration in gene expression p旭ays a significant ro旭e in mou旭ding 旭ife to the stressfu旭 intertida旭 旭ifesty旭es The gene expression profi旭es of N concinna from the intertida旭 and subtida旭 were changed on trans-

p旭antationp but the extent of this change depended on the habitat of origins The expression profi旭e of transp旭anted subtida旭 旭impets changed showing gradua旭 acc旭imation to the stressfu旭 intertida旭 zones In contrastp after a month spent at depths be旭ow ゲズ mp the expres-

sion profi旭e of the transp旭anted intertida旭 anima旭s 旭arge旭y remained the sames A旭though the metabo旭ism of Antarctic benthic species is much s旭ower than temperate anima旭s ｪPeckp ゴグゲ葦ｫp gene expression responses can be rapid with significant responses seen within an hourp as measured in heat shock experiments previous旭y performed on this species ｪC旭arkp Fraserp ｹ Peckp ゴググ芦ｫs Hencep the 旭ack or very s旭ow rate of change in gene expression profi旭es of the transp旭anted anima旭s was surprising and therefore other factors regu旭ating gene expression must be invo旭veds The subject of these experimentsp the Antarctic 旭impet is a broadcast spawner and thus has the advantage that such evaluations are made on the basis that the ecotypes sam-

p旭ed are from the same genetic popu旭ations Thereforep the differ-ences in expression profi旭es between intertida旭 and subtida旭 cohorts represented phenotypic p旭asticity and were not the resu旭t of genetic differentiationp as supported by current and previous genetic ana旭-yses ｪHoffman et a旭sp ゴグゲグｫs The intertida旭 and subtida旭 expression profi旭es were associated with differences in methy旭ation patternsp which diminished in the common garden experiment over ゾ months a旭ong with the dominating intertida旭 gene expression patterns These data strong旭y suggest that epigenetic regu旭ation ｪmethy旭ationｫ may

F IGURE ザ科Summary of gene expression resu旭tsp showing predominant functiona旭 groups in each transp旭anted group and shared transcripts compared with contro旭 subtida旭 anima旭ss For a fu旭旭 set of gene IDsp see tab旭es in Data Sジp Sズ and Tab旭es Sゲp Sゴ

科架 科 | 科ゲゾ芦ゾFunctional EcologyCLARK ET AL

p旭ay a significant ro旭e in shaping the physio旭ogy of these anima旭s to their particu旭ar habitat ｪintertida旭 or subtida旭ｫs

Physio旭ogica旭旭yp N concinna shows considerable modifications

according to habitat zone and depth ｪMor旭ey et a旭sp ゴグゲグq Wa旭旭er et a旭sp ゴググ葦q Weihe ｹ Abe旭ep ゴググ芦ｫs In addition to the pub旭ished datap further data are avai旭ab旭e showing that subtida旭 anima旭s have almost double the wet body mass compared with intertidal ani-

ma旭s ｪData Sゴｫ and that the therma旭 旭imits of intertida旭 anima旭s are highers The upper 旭etha旭 旭imit of anima旭s warmed at ゲﾀC｠hr was ザs芦ﾀC higher in intertida旭 anima旭s compared with those in the subtida旭 zone (H ゴザsズグp df ゲp p sググゲｫ ｪData Sゲｫs Thusp there is considerab旭e evidence that there are defined physio旭ogica旭 requirements for 旭ife in the two different zonesp a旭though there are c旭ear旭y dependency effectss For examp旭ep the righting abi旭ity of both forms is re旭ated to she旭旭 shapep with righting being more efficient in intertida旭 anima旭s ｪMor旭ey et a旭sp ゴグゲグｫs The intertida旭 anima旭s are a旭so better ab旭e to survive hypoxia as there is a bigger air pocket reserve in the ta旭旭er she旭旭s ｪWeihe ｹ Abe旭ep ゴググ芦ｫs Therma旭 to旭erance can be re旭ated to the different therma旭 histories of the two zonesp with intertida旭 ani-ma旭s regu旭ar旭y exposed to air temperatures in excess of the sha旭旭ow subtida旭 waters ｪC旭arkp Geiss旭erp et a旭sp ゴググ芦ｫs These data on 旭impets are va旭idated by physio旭ogica旭 differences found between intertida旭 and subtida旭 popu旭ations of other speciess For examp旭ep eva旭uations of c旭am and musse旭 species show that intertida旭 anima旭s exhibit met-abo旭ic rate depressionp higher rates of anaerobic metabo旭ismp abi旭ity

FIGURE ジ科 STRING output showing putative proteinprotein interactions for 9- month acclimated intertidal animals Transcripts

represented by red circ旭es are putative antioxidantsq green circ旭esr involved in the pentose shunt and carbohydrate metabolism

ye旭旭owr membrane transportersq b旭uer various functionsr GADゴ ｪneurotransmissionｫp ASNS ｪunfo旭ded protein responseｫp GLUL ｪg旭utamate metabo旭ismｫp MPOr ｪimmuneｫp FBLNゲ ｪce旭旭 adhesionｫs Transcripts invo旭ved in g旭utathiony旭ation are grouped in the b旭ack circle

Gene ID Gene name Putative function

ABCAゲ ATP binding cassette subfami旭y A member

Membrane transporter

AQPジ Aquaporin ジ Membrane transporter

ASNS Asparagine synthetase Invo旭ved in the Unfo旭ded Protein Response

CAT Catalase Antioxidant

FBLNゲ Fibu旭in Cell adhesion

GADゴ G旭utamate decarboxy旭ase ゴ Neurotransmission

GFPTゴ G旭utamine fructose 葦 phosphate transaminase 2

G旭ucose f旭ux into the hexosamine pathway

GLUL G旭utamate ammonia 旭igase G旭utamate metabo旭ism

GSTAゲ G旭utathione S- transferase G旭utathiony旭ationp antioxidant

GSTTゴB G旭utathione S- transferase G旭utathiony旭ationp antioxidant

HK1 Hexokinase ゲ G旭yco旭ysis

MGSTゴ Microsomal

g旭utathione S- transferase

G旭utathiony旭ationp antioxidant

MPO Mye旭operoxidase Immune

PGD Phosphog旭uconate dehydrogenase Pentose shunt pathway

SLCゲAゴ Solute carrier family 1 member Membrane transporter

SLCゼAズ Solute carrier family 7 member Membrane transporter

TALDOゲ Transaldolase Important for the balance of

metabolites in the pentose shunt

pathway

TABLE ザ科Gene identifiersp names and putative functions of transcripts involved

in the major network identified in the

STRING program for ゾ month acc旭imated intertidal animals

ゲゾゾグ科 |科 科架Functional Ecology CLARK ET AL

for air breathing and greater hypoxia to旭erance compared with their subtida旭 cohorts ｪA旭tierip ゴググ葦q Tag旭iaro旭op C旭avierp Chauvaudp Kokenp ｹ Gra旭旭p ゴグゲゴｫs There are a旭so differences between anima旭s in the two habitats with regard to she旭旭 morpho旭ogyp physio旭ogica旭 ener-getics and fatty acid profi旭es ｪFreitesp Labartap ｹ Fern史ndez Reir趣zp ゴググゴq Hinchp Bai旭eyp ｹ Greenp ゲゾ芦葦q Labartap Fern史ndez Reir趣zp ｹ Babarrop ゲゾゾゼｫs Howeverp to datep there has been 旭itt旭e mo旭ecu旭ar data underpinning these physio旭ogica旭 f旭exibi旭itiess

The data described here clearly show considerable differences

in 旭impet gene expression profi旭es re旭ated to zonations These dif-ferences can be assigned to biochemica旭 pathwaysp which corre旭ate with these different capacities and underpin the phenotypic differ-

encess The transp旭anted intertida旭 anima旭s in the subtida旭 zone main-

tained an expression profi旭e dominated by transcripts indicative of 旭ife in the intertida旭 where there is requirement to dea旭 with contin-

ua旭旭y f旭uctuating and stressfu旭 conditionsp such as emersionp high UV and a旭most constant buffeting with brash ices This was evidenced by upregu旭ated transcripts putative旭y invo旭ved in the responses to emersionp invo旭ving anoxia and hypoxia such as anoxia induced gr旭 旭ike protein and HYOUゲs Additiona旭旭yp genes invo旭ved in the c旭assica旭 stress response were present with genes putative旭y invo旭ved in an-

tioxidation and combating ROS ｪFigure ザp Tab旭e Sゲｫs These inc旭uded transcripts with high sequence simi旭arity to genes encoding antiox-

idants ｪGSRp GSTPゲp MGSTゴp NXNp SODゲ and SODゴｫ and the pen-

tose shunt ｪ葦 phosphog旭uconate dehydrogenase ｬPGDｭｫs The 旭atter is an important source of reducing ce旭旭u旭ar NADPH to combat ROSs Similar data were found in analyses of metabolic cycles in intertidal

musse旭s where 旭arge sca旭e changes in physio旭ogica旭 state and metab-

o旭ite profi旭es were associated with regu旭ar periods of hypoxia and the switch to anaerobic metabo旭ism associated with emersion ｪGracey ｹ Connorp ゴグゲ葦ｫs

The constant production of proteins that are considered pro-

tective in the intertida旭 anima旭s agrees with the previous identifi-cation of what has been termed preparative defence in Lottiap an intertida旭 gastropod ｪDongp Mi旭旭erp Sandersp ｹ Somerop ゴググ芦ｫ and wconstitutive front旭oadingx in heat resi旭ient popu旭ations of cora旭s ｪBarshis et a旭sp ゴグゲザｫs In the former studyp candidate heat shock proteins ｪHSPゼグｫ were measureds High constitutive expression 旭eve旭s of these genes were maintained as a pre emptive defence against extreme and unpredictab旭e heat stress in the intertida旭 zone ｪDong et a旭sp ゴググ芦ｫs Simi旭ar旭yp high 旭eve旭s of constitutive expression of the inducib旭e forms of HSPゼグ were a旭so demonstrated in N con-

cinna in response to tida旭 emersionp both natura旭旭y and artificia旭旭y induced ｪC旭ark ｹ Peckp ゴググゾq C旭arkp Geiss旭erp et a旭sp ゴググ芦ｫs In the cora旭 studyp RNA Seq ana旭yses revea旭ed that the more resi旭ient pop-

u旭ations of cora旭 expressed higher 旭eve旭s of heat shock proteins and antioxidant enzymes in addition to a series of genes invo旭ved in a wide range of functions such as apoptosis and immune responses ｪBarshis et a旭sp ゴグゲザｫs This constant production of defence genes is energetica旭旭y cost旭y and can resu旭t in ce旭旭u旭ar trade offs ｪSorensen ｹ Loeschckep ゴググ葦ｫs In the N concinna intertida旭 expression profi旭esp this increased energetic requirement may be ref旭ected in e旭evated levels of transcripts putatively involved in carbohydrate and fatty

acid metabo旭ism a旭ong with transcripts invo旭ved in transcription and trans旭ation ｪTab旭e Sザp Data Sゴｫs

Additiona旭 functiona旭 groupings were a旭so identified in the upreg-

u旭ated transcripts of the intertida旭 transp旭ants ｪTab旭e Sゴｫs The most common of these ｪゼ鯵 of the tota旭ｫ comprised cytoske旭eta旭 proteinss It has 旭ong been demonstrated that in yeast actin acts as an oxidative stress sensor ｪFarah ｹ Ambergp ゴググゼｫ a旭though an increasing number of environmenta旭 studies have identified a wider range of cytoske旭-eta旭 proteinsp inc旭uding the tubu旭ins and co旭旭agenp as identified here ｪFigure ザp Tab旭e Sゴｫs These cytoske旭eton genes have been shown to be upregu旭ated in response to different types of stressp such as tem-

perature in Ciona and Mytilus and elevated PCO2 in Crassostrea gigas

ｪFie旭dsp Zuzowp ｹ Tomanekp ゴグゲゴq Serafinip Hannp Ku旭tzp ｹ Tomanekp ゴグゲゲq Tomanekp Zuzowp Hittp Serafinip ｹ Va旭enzue旭ap ゴグゲゴｫs The data here add to an increasing body of evidence that sing旭e ce旭旭ed or-ganisms through to higher vertebrates have incorporated the cyto-

ske旭eton within numerous signa旭旭ing pathways and use the dynamic state of the cytoskeleton as an important indicator of cell health

ｪLeadsham ｹ Gour旭ayp ゴググ芦ｫs Transcriptiona旭 ana旭yses have showed co expression of antioxidants and cytoske旭eta旭 proteins indicating that the cytoske旭eton is a major target of ROS ｪLeadsham ｹ Gour旭ayp ゴググ芦ｫs Thusp upregu旭ation of cytoske旭eton transcripts can be indica-

tors of increased environmenta旭旭y induced ROS activitys A further modu旭e inc旭uded transcripts putative旭y invo旭ved in DNA damage and repair ｪザ鯵 of tota旭ｫ ｪFigure ザp Tab旭e Sゴｫs This is another documented response to environmenta旭 stress and ROS accumu旭ationp which can invo旭ve nuc旭ear fragmentation and DNA degradation ｪFarah ｹ Ambergp ゴググゼq Ga旭hardop Hastingsp ｹ Rosenbergp ゴググゼｫs Other tran-

scripts of note in the intertidal transplants included those putatively

invo旭ved in the she旭旭 matrixp such as thrombospondin and pro旭ine rich proteins ｪTab旭e Sゲｫp which is not entire旭y surprising as the inter-tidal animals have thicker shells compared with subtidal animals

Converse旭yp the subtida旭 anima旭s transp旭anted to the intertida旭 region changed their expression profi旭es towards that of intertida旭 anima旭s rather rapid旭y with an ana旭ysis of functiona旭 groupings re-

vea旭ing a simi旭ar profi旭e to the intertida旭 anima旭ss Overa旭旭p ゴ鯵 of tran-

scripts were putative旭y invo旭ved in DNA damage and repairp ジ鯵 in antioxidant activity and ゾ鯵 invo旭ved in the cytoske旭etons The 旭atter category inc旭uded ge旭so旭inp an actin depo旭ymerising protein which has been imp旭icated in signa旭旭ing processes ｪFarah ｹ Ambergp ゴググゼｫ ｪTab旭e Sゴｫs A旭so present were transcripts putative旭y invo旭ved in the pentose shunt ｪPGD which provides NADPH intermediates for com-

bating ROSｫ and the she旭旭 matrix ｪchitin synthase and carbonic anhy-

draseｫs Interesting旭yp ゲザ annotated transcripts were shared between the subtidal and intertidal transplanted animals when their respec-

tive expression profi旭es were compared ｪFigure ザｫs These coded for a wide range of functions inc旭uding the pentose shunt ｪPGDｫp DNA re-

pair ｪLIGゲｫ and protein fo旭ding and degradation ｪPPIBp RANBPゴｫs Of particu旭ar notep was SORBSゴp which assemb旭es signa旭旭ing comp旭exesp acting as a 旭ink between kinasesp such as PAKゲ ｪa旭so present in these annotationsｫp with the cytoske旭etonp again high旭ighting the potentia旭 importance of the cytoske旭eton in environmenta旭 responsess Thusp the transp旭anted subtida旭 anima旭s showed an expression profi旭e

科架 科 | 科ゲゾゾゲFunctional EcologyCLARK ET AL

moving towards that of an intertida旭 anima旭p indicative of an increas-

ing requirement to combat ROS and produce a thicker she旭旭 for de-

fence against brash ice in the intertida旭 regions This a旭so indicates that the environmenta旭 stresses in the intertida旭 zone ｪhypoxiap des-

iccationp temperaturep icep etcsｫ act as strong environmenta旭 cues on ce旭旭u旭ar expression patternss

These mo旭ecu旭ar findings in N concinnap which underpin the physio旭ogica旭 responses to the intertida旭 zone are seen in other spe-

ciesp in particu旭ar when those species are subjected to transp旭an-

tation experimentss For examp旭ep compensation of the heat shock response occurred in mussels moved between the intertidal and

subtida旭 zones ｪHa旭pinp Mengep ｹ Hofmannp ゴググジｫ and the transcrip-

tiona旭 and metabo旭omic cyc旭ic response to hypoxia in intertida旭 mus-

sels was still observed when individuals of M californianus were kept

submerged for ゴ weekss These musse旭s continued to undergo spon-

taneous bouts of anaerobiosis to mimic the conditions of emersionp as they wou旭d experience in the intertida旭 zone ｪGracey ｹ Connorp ゴグゲ葦ｫs Whi旭e the anima旭s in some transp旭ant experiments showed comp旭ete compensation of physio旭ogy after transp旭antation to a dif-ferent shore zone ｪA旭tierip ゴググ葦ｫp other studies recorded 旭ags in com-

pensation whichp it was suggestedp were due to either differences in weco旭ogica旭 memoryx or wadaptation de旭ayx ｪFreites et a旭sp ゴググゴq Labarta et a旭sp ゲゾゾゼｫs This wde旭ayx was a旭most certain旭y due to the time sca旭es of the experimentsp as even in the temperate M gallo-

provincialis ザ葦ズグ days were required for the anima旭s to adjust their physio旭ogy to that of a different habitat ｪrocky shore vss subtida旭ｫ ｪFreites et a旭sp ゴググゴｫs

This delay was also seen in the molecular data described here

as there was a distinctive and persistent expression profi旭e associ-ated with 旭ife in the intertida旭p even when intertida旭 anima旭s were transp旭anted to the subtida旭 zones The most parsimonious exp旭ana-

tions for these data are either that it took the anima旭s a 旭ong time to use their physio旭ogica旭 f旭exibi旭ity and acc旭imate to their new condi-tions as has been demonstrated for Antarctic marine invertebrates in their therma旭 physio旭ogy ｪPeckp Mor旭eyp Richardp ｹ C旭arkp ゴグゲジｫp or that the expression profi旭es associated with the intertida旭 region had become fixed in some ways The 旭atter may potentia旭旭y act via epigenetic factorsp one of which is methy旭ations Hencep a common garden experiment was performed a旭ongside an eva旭uation of ge-

nome methylation patterns

After being he旭d for ゾ months in identica旭 aquarium condi-tionsp there were sti旭旭 some significant differences in the gene expression profi旭es of the intertida旭 and subtida旭 cohortss This was surprisingp given the time sca旭e which was more than suffi-cient to enab旭e fu旭旭 physio旭ogica旭 acc旭imation in Antarctic species ｪMor旭ey et a旭sp ゴグゲゲq Peck et a旭sp ゴグゲジｫp and thereforep harmonisa-

tion of the transcription profi旭es of both cohorts might have been expecteds A旭though the STRING program showed no significant networks in the subtida旭 anima旭sp there was sti旭旭 a sma旭旭 core of ゲゼ genes enriched in the intertida旭 acc旭imated anima旭s ｪFigure ジｫp which was c旭ear旭y a remnant from the very extensive gene ex-

pression differences previously identified in the transplant

experiment ｪData Sジ and Sズｫs A critica旭 core of an antioxidant

response remainedp comprising severa旭 wc旭assica旭x stress re-

sponse genesp in particu旭ar severa旭 members of the g旭utathione S transferase fami旭yp which are invo旭ved in protecting against ROSs A旭so present were genes within the pentose shunt path-

way ｪPGDp SLCゼDズｫp which generate reducing equiva旭ents in the form of NADPHp again invo旭ved in preventing oxidative stress ｪFigure ジｫs Hencep the ce旭旭u旭ar pathways invo旭ved in combating ROS remained as the 旭ast set of genes to have their regu旭ation pattern erased in these acc旭imated anima旭sp thus indicating the importance of antioxidant protection systems in the intertida旭 zones Whi旭e some proteinsp such as PGD identified in this studyp have been shown to be redox sensitive ｪWang et a旭sp ゴグゲゴｫp our acc旭imation study indicates that upregu旭ation of these tran-

scripts was not a rapid response to a potentia旭 stressp but more 旭ike旭y a durab旭e preventive mechanism to the intertida旭 旭ifesty旭ep simi旭ar to the preparative defence and constitutive front旭oading described ear旭ier ｪBarshis et a旭sp ゴグゲザq Dong et a旭sp ゴググ芦ｫs It may be that these remnants of intertida旭 gene expression are the re-

su旭t of irreversib旭e deve旭opmenta旭 p旭asticityp but c旭ear旭y 旭onger common garden experiments a旭ong with expression profi旭ing of new旭y deve旭oping 旭arvae wou旭d be required to detect thiss Thusp within the context of the current experimentp the question arose as to how these genes are tagged and epigenetics presented as a prime candidate for investigations

Epigenetic changes to genomes are increasing旭y recognised as an important factor in species modification to local environmental

conditions ｪBossdorf et a旭sp ゴググ芦ｫs The mechanisms behind such changes inc旭ude methy旭ated cytosine residues ｪズ methy旭cytosine or ズmCｫq the remode旭旭ing of chromatin structure through chemi-ca旭 changes to histone proteins and regu旭ation by sma旭旭 RNA mo旭-ecu旭ess These mechanisms are not mutua旭旭y exc旭usive and may combine to act in a comp旭ex manner ｪBossdorf et a旭sp ゴググ芦ｫs The best studied of these is ズmCs A re旭ative旭y simp旭e test of differen-

tia旭 cytosine methy旭ation between different popu旭ationsp cohorts or 旭ife history traits is the use of MSAP ana旭ysis ｪReyna Lopezp Simpsonp ｹ RuizHerrerap ゲゾゾゼq Sun et a旭sp ゴグゲジｫs Whi旭e methy旭-ated cytosines have been identified in a number of invertebrates

ｪTweediep Char旭tonp C旭arkp ｹ Birdp ゲゾゾゼｫp to date 旭itt旭e is known about how methy旭ation affects gene expression in these taxas The data here showed significant differences in methy旭ation patterns between subtida旭 and intertida旭 anima旭sp both at the start of the experiment and between the transp旭anted individua旭sp indicating that epigenetic imprinting p旭ays an important ro旭e in the differenti-ation of N concinna ecotypes to their respective habitats This pat-

ternp howeverp was not observed in the individua旭s samp旭ed at the end of the acc旭imation experiment indicating that the methy旭ation may be transient It should be noted that in this preliminary trial a

very restricted proportion of the genome was assayed for methy旭-ation and therefore methylation levels were almost certainly more

extensive than demonstrated heres These intriguing data demon-

strate the c旭ear need for more extensive studies especia旭旭y as a sma旭旭 core of wintertida旭 profi旭ex genes remained upregu旭ated after ゾ monthss These resu旭ts showing transient methy旭ation status are

ゲゾゾゴ科 |科 科架Functional Ecology CLARK ET AL

a旭so simi旭ar to those in other speciesp for examp旭e in trout where sa旭t enriched diets can trigger short term genome wide methy旭-ation differences ｪMoranp Marco Riusp Megiasp Cove旭o Sotop ｹ Perez Figueroap ゴグゲザｫ and she旭旭fish in which methy旭ation is sug-

gested to act as an immune regu旭atory factor ｪShangp Sup Wanp ｹ Sup ゴグゲズｫs Whi旭e in this experimentp it was not possib旭e to assign methy旭ation to specific genesp there was a corre旭ation between the extent of methy旭ation status and the difference in gene expression profiles associated with a particular environment The methylation

旭eve旭s decreased in the common garden experimentp where there was a complete absence of the natural environmental cues further

substantiating the methy旭ationr habitat corre旭ationss

ズ科 |科CONCLUSIONS

These datap using RNA Seq revea旭 the under旭ying comp旭exity of response to maintaining 旭ife in the intertida旭p beyond previous can-

didate gene approachess These profi旭es indicated that e旭evated expression of genes associated with antioxidant responses form a constitutive defence against the harsh intertida旭 habitats Whi旭e the core of such a response is durab旭e 旭asting many months after the en-

vironmenta旭 cue has been removed it may be reversib旭es Significant differences existed in the methy旭ation patterns between intertida旭 and subtida旭 anima旭sp which were reduced during the common gar-den experimentp indicating that epigenetic factors may inf旭uence the response to habitats These data demonstrating significant pheno-

typic and methy旭ation p旭asticity within a genetica旭旭y homogeneous popu旭ation are high旭y re旭evant to g旭oba旭 eva旭uations of species abi旭i-ties to respond to specific habitats and to c旭imate changes C旭ear旭yp 旭inking 旭eve旭s of methy旭ation and｠or other epigenetic factorsp such as histone acety旭ation and microRNAs to specific genes and functions will provide critical data towards a more comprehensive mechanis-

tic understanding of species ce旭旭u旭ar resi旭ience under future c旭imate changes

ACKNOWLEDG EMENTS

This project was 旭arge旭y financed by Natura旭 Environment Research Counci旭 ｪNERCｫ core funding to BASs The authors thank a旭旭 members of the Rothera dive team for providing samp旭es and he旭ping with the intertida旭 co旭旭ections and Jamie O旭iver ｪBASｫ for creating Figures ゲ and ザs Overa旭旭p BAS diving was supported by the NERC Nationa旭 Faci旭ity for Scientific Diving at Obans MsSsCs and LsSsPs thank both Terri Souster for the donation of her nail varnish collection and

A旭an Hi旭旭 for his f旭y tipping paint for the 旭impet painting as part of the transp旭ant experiments AFLP ana旭ysis of methy旭ation patterns ｪMSAPｫ was performed at the NERC Biomo旭ecu旭ar Ana旭ysis Faci旭ity ｪNBAFｫ at Sheffie旭dp financed by a NERC UK access grant ｪNBAF芦ジゴｫ awarded to MsSsCs We thank Rache旭 Tucker for providing 旭abora-

tory assistance and Maria E旭ena Mannare旭旭i for providing assistancep training and guidance during the 旭aboratory extractions and 旭abe旭旭ing for the MSAP experiments

AUTHORSv CONTRIBUTIONS

MsSsCsp LsSsPs and JsIsHs conceived the studyq MsSsCs and LsSsPs per-formed the fie旭d work and co旭旭ected samp旭esq MsSsCsp JsIsHs and MsKs processed the samp旭esq MsKs and HsHs carried out the MSAP ana旭y-

sesq MsAsSsTs performed the bioinformatics ana旭ysesq MsSsCs was responsib旭e for the bio旭ogica旭 annotation and interpretationq MsSsCs wrote the paper with substantia旭 input from LsSsPs and JsIsHs

DATA ACCE SSIBILIT Y

A旭旭 sequencing data were submitted to the NCBI SRA with the Accession Numberr SRPグズゾズジグs

ORCID

Melody S Clark httpr｠｠orcidsorg｠グググググググゴザジジゴザ芦ゴジ

R E FE R E N CE S

A旭tierip As Hs ｪゴググ葦ｫs Inducib旭e variation in hypoxia to旭erance across the intertidal- subtidal distribution of the blue mussel Mytilus

edulis Marine Ecology Progress Seriesp 325p ゴゾズザググs httpsr｠｠doisorg｠ゲグsザザズジ｠mepsザゴズゴゾズ

Barshisp Ds Jsp Ladnerp Js Tsp O旭iverp Ts Asp Senecap Fs Osp Tray旭orKnow旭esp Nsp ｹ Pa旭umbip Ss Rs ｪゴグゲザｫs Genomic basis for cora旭 resi旭ience to c旭i-mate changes Proceedings of the National Academy of Sciences of the

United States of Americap 110p ゲザ芦ゼゲザゾゴs httpsr｠｠doisorg｠ゲグsゲグゼザ｠pnas1210224110

Bensonp Ds Asp KarschMizrachip Isp Lipmanp Ds Jsp Oste旭旭p Jsp ｹ Whee旭erp Ds Ls ｪゴググゼｫs GenBanks Nucleic Acids Researchp 35p DゴゲDゴズs httpsr｠｠doisorg｠ゲグsゲグゾザ｠nar｠gk旭ゾ芦葦

Bossdorfp Osp Richardsp Cs Lsp ｹ Pig旭iuccip Ms ｪゴググ芦ｫs Epigenetics for eco旭-ogistss Ecology Lettersp 11p ゲグ葦ゲゲズs

Chapmanp Rs Wsp Manciap Asp Bea旭p Msp Ve旭osop Asp Rathburnp Csp B旭airp Asp t Wirthp Es Fs ｪゴグゲゲｫs The transcriptomic responses of the eastern oysterp Crassostrea virginicap to environmenta旭 conditionss Molecular

Ecologyp 20p ゲジザゲゲジジゾs httpsr｠｠doisorg｠ゲグsゲゲゲゲ｠jsゲザ葦ズゴゾジXsゴグゲゲs グズグゲ芦sx

C旭arkp Ms Ssp Fraserp Ks Ps Psp ｹ Peckp Ls Ss ｪゴググ芦ｫs Antarctic marine mo旭旭uscs do have an HSPゼグ heat shock responses Cell Stress and Chaperonesp 13p ザゾジゾs httpsr｠｠doisorg｠ゲグsゲググゼ｠sゲゴゲゾゴググ芦ググゲジ芦

C旭arkp Ms Ssp Geiss旭erp Psp Wa旭旭erp Csp Fraserp Ks Ps Psp Barnesp Ds Ks Asp ｹ Peckp Ls Ss ｪゴググ芦ｫs Low heat shock thresho旭ds in wi旭d Antarctic inter tidal limpets (Nacella concinnaｫs Cell Stress and Chaperonesp 13p ズゲズ芦s httpsr｠｠doisorg｠ゲグsゲググゼ｠sゲゴゲゾゴググ芦ググゲズゼ

C旭arkp Ms Ssp Husmannp Gsp Thornep Ms As Ssp Burnsp Gsp Truebanop Msp Peckp Ls Ssp t Phi旭ippp Es Es ｪゴグゲザｫs Hypoxia impacts 旭arge adu旭ts firstr Consequences in a warming wor旭ds Global Change Biologyp 19p ゴゴズゲゴゴ葦ザs httpsr｠｠doisorg｠ゲグsゲゲゲゲ｠gcbsゲゴゲゾゼ

C旭arkp Ms Ssp ｹ Peckp Ls Ss ｪゴググゾｫs Triggers of the HSPゼグ stress responser Environmenta旭 responses and 旭aboratory manipu旭ation in an Antarctic marine invertebrate (Nacella concinnaｫs Cell Stress and Chaperonesp 14p 葦ジゾ葦葦グs httpsr｠｠doisorg｠ゲグsゲググゼ｠sゲゴゲゾゴググゾグゲゲゼx

De Wo旭fp Hsp Backe旭jaup Tsp ｹ Verhagenp Rs ｪゲゾゾ芦ｫs Spatio tempora旭 ge-

netic structure and gene f旭ow between two distinct she旭旭 morphs of the p旭anktonic deve旭oping periwink旭e Littorina striata (Mollusca

Prosobranchiaｫs Marine Ecology Progress Seriesp 163p ゲズズゲ葦ザs httpsr｠｠doisorg｠ゲグsザザズジ｠mepsゲ葦ザゲズズ

Dongp Ys Wsp Mi旭旭erp Ls Psp Sandersp Js Gsp ｹ Somerop Gs Ns ｪゴググ芦ｫs Heat shock protein ゼグ ｪHspゼグｫ expression in four 旭impets of the genus

科架 科 | 科ゲゾ芦ゾFunctional EcologyCLARK ET AL

p旭ay a significant ro旭e in shaping the physio旭ogy of these anima旭s to their particu旭ar habitat ｪintertida旭 or subtida旭ｫs

Physio旭ogica旭旭yp N concinna shows considerable modifications

according to habitat zone and depth ｪMor旭ey et a旭sp ゴグゲグq Wa旭旭er et a旭sp ゴググ葦q Weihe ｹ Abe旭ep ゴググ芦ｫs In addition to the pub旭ished datap further data are avai旭ab旭e showing that subtida旭 anima旭s have almost double the wet body mass compared with intertidal ani-

ma旭s ｪData Sゴｫ and that the therma旭 旭imits of intertida旭 anima旭s are highers The upper 旭etha旭 旭imit of anima旭s warmed at ゲﾀC｠hr was ザs芦ﾀC higher in intertida旭 anima旭s compared with those in the subtida旭 zone (H ゴザsズグp df ゲp p sググゲｫ ｪData Sゲｫs Thusp there is considerab旭e evidence that there are defined physio旭ogica旭 requirements for 旭ife in the two different zonesp a旭though there are c旭ear旭y dependency effectss For examp旭ep the righting abi旭ity of both forms is re旭ated to she旭旭 shapep with righting being more efficient in intertida旭 anima旭s ｪMor旭ey et a旭sp ゴグゲグｫs The intertida旭 anima旭s are a旭so better ab旭e to survive hypoxia as there is a bigger air pocket reserve in the ta旭旭er she旭旭s ｪWeihe ｹ Abe旭ep ゴググ芦ｫs Therma旭 to旭erance can be re旭ated to the different therma旭 histories of the two zonesp with intertida旭 ani-ma旭s regu旭ar旭y exposed to air temperatures in excess of the sha旭旭ow subtida旭 waters ｪC旭arkp Geiss旭erp et a旭sp ゴググ芦ｫs These data on 旭impets are va旭idated by physio旭ogica旭 differences found between intertida旭 and subtida旭 popu旭ations of other speciess For examp旭ep eva旭uations of c旭am and musse旭 species show that intertida旭 anima旭s exhibit met-abo旭ic rate depressionp higher rates of anaerobic metabo旭ismp abi旭ity

FIGURE ジ科 STRING output showing putative proteinprotein interactions for 9- month acclimated intertidal animals Transcripts

represented by red circ旭es are putative antioxidantsq green circ旭esr involved in the pentose shunt and carbohydrate metabolism

ye旭旭owr membrane transportersq b旭uer various functionsr GADゴ ｪneurotransmissionｫp ASNS ｪunfo旭ded protein responseｫp GLUL ｪg旭utamate metabo旭ismｫp MPOr ｪimmuneｫp FBLNゲ ｪce旭旭 adhesionｫs Transcripts invo旭ved in g旭utathiony旭ation are grouped in the b旭ack circle

Gene ID Gene name Putative function

ABCAゲ ATP binding cassette subfami旭y A member

Membrane transporter

AQPジ Aquaporin ジ Membrane transporter

ASNS Asparagine synthetase Invo旭ved in the Unfo旭ded Protein Response

CAT Catalase Antioxidant

FBLNゲ Fibu旭in Cell adhesion

GADゴ G旭utamate decarboxy旭ase ゴ Neurotransmission

GFPTゴ G旭utamine fructose 葦 phosphate transaminase 2

G旭ucose f旭ux into the hexosamine pathway

GLUL G旭utamate ammonia 旭igase G旭utamate metabo旭ism

GSTAゲ G旭utathione S- transferase G旭utathiony旭ationp antioxidant

GSTTゴB G旭utathione S- transferase G旭utathiony旭ationp antioxidant

HK1 Hexokinase ゲ G旭yco旭ysis

MGSTゴ Microsomal

g旭utathione S- transferase

G旭utathiony旭ationp antioxidant

MPO Mye旭operoxidase Immune

PGD Phosphog旭uconate dehydrogenase Pentose shunt pathway

SLCゲAゴ Solute carrier family 1 member Membrane transporter

SLCゼAズ Solute carrier family 7 member Membrane transporter

TALDOゲ Transaldolase Important for the balance of

metabolites in the pentose shunt

pathway

TABLE ザ科Gene identifiersp names and putative functions of transcripts involved

in the major network identified in the

STRING program for ゾ month acc旭imated intertidal animals

ゲゾゾグ科 |科 科架Functional Ecology CLARK ET AL

for air breathing and greater hypoxia to旭erance compared with their subtida旭 cohorts ｪA旭tierip ゴググ葦q Tag旭iaro旭op C旭avierp Chauvaudp Kokenp ｹ Gra旭旭p ゴグゲゴｫs There are a旭so differences between anima旭s in the two habitats with regard to she旭旭 morpho旭ogyp physio旭ogica旭 ener-getics and fatty acid profi旭es ｪFreitesp Labartap ｹ Fern史ndez Reir趣zp ゴググゴq Hinchp Bai旭eyp ｹ Greenp ゲゾ芦葦q Labartap Fern史ndez Reir趣zp ｹ Babarrop ゲゾゾゼｫs Howeverp to datep there has been 旭itt旭e mo旭ecu旭ar data underpinning these physio旭ogica旭 f旭exibi旭itiess

The data described here clearly show considerable differences

in 旭impet gene expression profi旭es re旭ated to zonations These dif-ferences can be assigned to biochemica旭 pathwaysp which corre旭ate with these different capacities and underpin the phenotypic differ-

encess The transp旭anted intertida旭 anima旭s in the subtida旭 zone main-

tained an expression profi旭e dominated by transcripts indicative of 旭ife in the intertida旭 where there is requirement to dea旭 with contin-

ua旭旭y f旭uctuating and stressfu旭 conditionsp such as emersionp high UV and a旭most constant buffeting with brash ices This was evidenced by upregu旭ated transcripts putative旭y invo旭ved in the responses to emersionp invo旭ving anoxia and hypoxia such as anoxia induced gr旭 旭ike protein and HYOUゲs Additiona旭旭yp genes invo旭ved in the c旭assica旭 stress response were present with genes putative旭y invo旭ved in an-

tioxidation and combating ROS ｪFigure ザp Tab旭e Sゲｫs These inc旭uded transcripts with high sequence simi旭arity to genes encoding antiox-

idants ｪGSRp GSTPゲp MGSTゴp NXNp SODゲ and SODゴｫ and the pen-

tose shunt ｪ葦 phosphog旭uconate dehydrogenase ｬPGDｭｫs The 旭atter is an important source of reducing ce旭旭u旭ar NADPH to combat ROSs Similar data were found in analyses of metabolic cycles in intertidal

musse旭s where 旭arge sca旭e changes in physio旭ogica旭 state and metab-

o旭ite profi旭es were associated with regu旭ar periods of hypoxia and the switch to anaerobic metabo旭ism associated with emersion ｪGracey ｹ Connorp ゴグゲ葦ｫs

The constant production of proteins that are considered pro-

tective in the intertida旭 anima旭s agrees with the previous identifi-cation of what has been termed preparative defence in Lottiap an intertida旭 gastropod ｪDongp Mi旭旭erp Sandersp ｹ Somerop ゴググ芦ｫ and wconstitutive front旭oadingx in heat resi旭ient popu旭ations of cora旭s ｪBarshis et a旭sp ゴグゲザｫs In the former studyp candidate heat shock proteins ｪHSPゼグｫ were measureds High constitutive expression 旭eve旭s of these genes were maintained as a pre emptive defence against extreme and unpredictab旭e heat stress in the intertida旭 zone ｪDong et a旭sp ゴググ芦ｫs Simi旭ar旭yp high 旭eve旭s of constitutive expression of the inducib旭e forms of HSPゼグ were a旭so demonstrated in N con-

cinna in response to tida旭 emersionp both natura旭旭y and artificia旭旭y induced ｪC旭ark ｹ Peckp ゴググゾq C旭arkp Geiss旭erp et a旭sp ゴググ芦ｫs In the cora旭 studyp RNA Seq ana旭yses revea旭ed that the more resi旭ient pop-

u旭ations of cora旭 expressed higher 旭eve旭s of heat shock proteins and antioxidant enzymes in addition to a series of genes invo旭ved in a wide range of functions such as apoptosis and immune responses ｪBarshis et a旭sp ゴグゲザｫs This constant production of defence genes is energetica旭旭y cost旭y and can resu旭t in ce旭旭u旭ar trade offs ｪSorensen ｹ Loeschckep ゴググ葦ｫs In the N concinna intertida旭 expression profi旭esp this increased energetic requirement may be ref旭ected in e旭evated levels of transcripts putatively involved in carbohydrate and fatty

acid metabo旭ism a旭ong with transcripts invo旭ved in transcription and trans旭ation ｪTab旭e Sザp Data Sゴｫs

Additiona旭 functiona旭 groupings were a旭so identified in the upreg-

u旭ated transcripts of the intertida旭 transp旭ants ｪTab旭e Sゴｫs The most common of these ｪゼ鯵 of the tota旭ｫ comprised cytoske旭eta旭 proteinss It has 旭ong been demonstrated that in yeast actin acts as an oxidative stress sensor ｪFarah ｹ Ambergp ゴググゼｫ a旭though an increasing number of environmenta旭 studies have identified a wider range of cytoske旭-eta旭 proteinsp inc旭uding the tubu旭ins and co旭旭agenp as identified here ｪFigure ザp Tab旭e Sゴｫs These cytoske旭eton genes have been shown to be upregu旭ated in response to different types of stressp such as tem-

perature in Ciona and Mytilus and elevated PCO2 in Crassostrea gigas

ｪFie旭dsp Zuzowp ｹ Tomanekp ゴグゲゴq Serafinip Hannp Ku旭tzp ｹ Tomanekp ゴグゲゲq Tomanekp Zuzowp Hittp Serafinip ｹ Va旭enzue旭ap ゴグゲゴｫs The data here add to an increasing body of evidence that sing旭e ce旭旭ed or-ganisms through to higher vertebrates have incorporated the cyto-

ske旭eton within numerous signa旭旭ing pathways and use the dynamic state of the cytoskeleton as an important indicator of cell health

ｪLeadsham ｹ Gour旭ayp ゴググ芦ｫs Transcriptiona旭 ana旭yses have showed co expression of antioxidants and cytoske旭eta旭 proteins indicating that the cytoske旭eton is a major target of ROS ｪLeadsham ｹ Gour旭ayp ゴググ芦ｫs Thusp upregu旭ation of cytoske旭eton transcripts can be indica-

tors of increased environmenta旭旭y induced ROS activitys A further modu旭e inc旭uded transcripts putative旭y invo旭ved in DNA damage and repair ｪザ鯵 of tota旭ｫ ｪFigure ザp Tab旭e Sゴｫs This is another documented response to environmenta旭 stress and ROS accumu旭ationp which can invo旭ve nuc旭ear fragmentation and DNA degradation ｪFarah ｹ Ambergp ゴググゼq Ga旭hardop Hastingsp ｹ Rosenbergp ゴググゼｫs Other tran-

scripts of note in the intertidal transplants included those putatively

invo旭ved in the she旭旭 matrixp such as thrombospondin and pro旭ine rich proteins ｪTab旭e Sゲｫp which is not entire旭y surprising as the inter-tidal animals have thicker shells compared with subtidal animals

Converse旭yp the subtida旭 anima旭s transp旭anted to the intertida旭 region changed their expression profi旭es towards that of intertida旭 anima旭s rather rapid旭y with an ana旭ysis of functiona旭 groupings re-

vea旭ing a simi旭ar profi旭e to the intertida旭 anima旭ss Overa旭旭p ゴ鯵 of tran-

scripts were putative旭y invo旭ved in DNA damage and repairp ジ鯵 in antioxidant activity and ゾ鯵 invo旭ved in the cytoske旭etons The 旭atter category inc旭uded ge旭so旭inp an actin depo旭ymerising protein which has been imp旭icated in signa旭旭ing processes ｪFarah ｹ Ambergp ゴググゼｫ ｪTab旭e Sゴｫs A旭so present were transcripts putative旭y invo旭ved in the pentose shunt ｪPGD which provides NADPH intermediates for com-

bating ROSｫ and the she旭旭 matrix ｪchitin synthase and carbonic anhy-

draseｫs Interesting旭yp ゲザ annotated transcripts were shared between the subtidal and intertidal transplanted animals when their respec-

tive expression profi旭es were compared ｪFigure ザｫs These coded for a wide range of functions inc旭uding the pentose shunt ｪPGDｫp DNA re-

pair ｪLIGゲｫ and protein fo旭ding and degradation ｪPPIBp RANBPゴｫs Of particu旭ar notep was SORBSゴp which assemb旭es signa旭旭ing comp旭exesp acting as a 旭ink between kinasesp such as PAKゲ ｪa旭so present in these annotationsｫp with the cytoske旭etonp again high旭ighting the potentia旭 importance of the cytoske旭eton in environmenta旭 responsess Thusp the transp旭anted subtida旭 anima旭s showed an expression profi旭e

科架 科 | 科ゲゾゾゲFunctional EcologyCLARK ET AL

moving towards that of an intertida旭 anima旭p indicative of an increas-

ing requirement to combat ROS and produce a thicker she旭旭 for de-

fence against brash ice in the intertida旭 regions This a旭so indicates that the environmenta旭 stresses in the intertida旭 zone ｪhypoxiap des-

iccationp temperaturep icep etcsｫ act as strong environmenta旭 cues on ce旭旭u旭ar expression patternss

These mo旭ecu旭ar findings in N concinnap which underpin the physio旭ogica旭 responses to the intertida旭 zone are seen in other spe-

ciesp in particu旭ar when those species are subjected to transp旭an-

tation experimentss For examp旭ep compensation of the heat shock response occurred in mussels moved between the intertidal and

subtida旭 zones ｪHa旭pinp Mengep ｹ Hofmannp ゴググジｫ and the transcrip-

tiona旭 and metabo旭omic cyc旭ic response to hypoxia in intertida旭 mus-

sels was still observed when individuals of M californianus were kept

submerged for ゴ weekss These musse旭s continued to undergo spon-

taneous bouts of anaerobiosis to mimic the conditions of emersionp as they wou旭d experience in the intertida旭 zone ｪGracey ｹ Connorp ゴグゲ葦ｫs Whi旭e the anima旭s in some transp旭ant experiments showed comp旭ete compensation of physio旭ogy after transp旭antation to a dif-ferent shore zone ｪA旭tierip ゴググ葦ｫp other studies recorded 旭ags in com-

pensation whichp it was suggestedp were due to either differences in weco旭ogica旭 memoryx or wadaptation de旭ayx ｪFreites et a旭sp ゴググゴq Labarta et a旭sp ゲゾゾゼｫs This wde旭ayx was a旭most certain旭y due to the time sca旭es of the experimentsp as even in the temperate M gallo-

provincialis ザ葦ズグ days were required for the anima旭s to adjust their physio旭ogy to that of a different habitat ｪrocky shore vss subtida旭ｫ ｪFreites et a旭sp ゴググゴｫs

This delay was also seen in the molecular data described here

as there was a distinctive and persistent expression profi旭e associ-ated with 旭ife in the intertida旭p even when intertida旭 anima旭s were transp旭anted to the subtida旭 zones The most parsimonious exp旭ana-

tions for these data are either that it took the anima旭s a 旭ong time to use their physio旭ogica旭 f旭exibi旭ity and acc旭imate to their new condi-tions as has been demonstrated for Antarctic marine invertebrates in their therma旭 physio旭ogy ｪPeckp Mor旭eyp Richardp ｹ C旭arkp ゴグゲジｫp or that the expression profi旭es associated with the intertida旭 region had become fixed in some ways The 旭atter may potentia旭旭y act via epigenetic factorsp one of which is methy旭ations Hencep a common garden experiment was performed a旭ongside an eva旭uation of ge-

nome methylation patterns

After being he旭d for ゾ months in identica旭 aquarium condi-tionsp there were sti旭旭 some significant differences in the gene expression profi旭es of the intertida旭 and subtida旭 cohortss This was surprisingp given the time sca旭e which was more than suffi-cient to enab旭e fu旭旭 physio旭ogica旭 acc旭imation in Antarctic species ｪMor旭ey et a旭sp ゴグゲゲq Peck et a旭sp ゴグゲジｫp and thereforep harmonisa-

tion of the transcription profi旭es of both cohorts might have been expecteds A旭though the STRING program showed no significant networks in the subtida旭 anima旭sp there was sti旭旭 a sma旭旭 core of ゲゼ genes enriched in the intertida旭 acc旭imated anima旭s ｪFigure ジｫp which was c旭ear旭y a remnant from the very extensive gene ex-

pression differences previously identified in the transplant

experiment ｪData Sジ and Sズｫs A critica旭 core of an antioxidant

response remainedp comprising severa旭 wc旭assica旭x stress re-

sponse genesp in particu旭ar severa旭 members of the g旭utathione S transferase fami旭yp which are invo旭ved in protecting against ROSs A旭so present were genes within the pentose shunt path-

way ｪPGDp SLCゼDズｫp which generate reducing equiva旭ents in the form of NADPHp again invo旭ved in preventing oxidative stress ｪFigure ジｫs Hencep the ce旭旭u旭ar pathways invo旭ved in combating ROS remained as the 旭ast set of genes to have their regu旭ation pattern erased in these acc旭imated anima旭sp thus indicating the importance of antioxidant protection systems in the intertida旭 zones Whi旭e some proteinsp such as PGD identified in this studyp have been shown to be redox sensitive ｪWang et a旭sp ゴグゲゴｫp our acc旭imation study indicates that upregu旭ation of these tran-

scripts was not a rapid response to a potentia旭 stressp but more 旭ike旭y a durab旭e preventive mechanism to the intertida旭 旭ifesty旭ep simi旭ar to the preparative defence and constitutive front旭oading described ear旭ier ｪBarshis et a旭sp ゴグゲザq Dong et a旭sp ゴググ芦ｫs It may be that these remnants of intertida旭 gene expression are the re-

su旭t of irreversib旭e deve旭opmenta旭 p旭asticityp but c旭ear旭y 旭onger common garden experiments a旭ong with expression profi旭ing of new旭y deve旭oping 旭arvae wou旭d be required to detect thiss Thusp within the context of the current experimentp the question arose as to how these genes are tagged and epigenetics presented as a prime candidate for investigations

Epigenetic changes to genomes are increasing旭y recognised as an important factor in species modification to local environmental

conditions ｪBossdorf et a旭sp ゴググ芦ｫs The mechanisms behind such changes inc旭ude methy旭ated cytosine residues ｪズ methy旭cytosine or ズmCｫq the remode旭旭ing of chromatin structure through chemi-ca旭 changes to histone proteins and regu旭ation by sma旭旭 RNA mo旭-ecu旭ess These mechanisms are not mutua旭旭y exc旭usive and may combine to act in a comp旭ex manner ｪBossdorf et a旭sp ゴググ芦ｫs The best studied of these is ズmCs A re旭ative旭y simp旭e test of differen-

tia旭 cytosine methy旭ation between different popu旭ationsp cohorts or 旭ife history traits is the use of MSAP ana旭ysis ｪReyna Lopezp Simpsonp ｹ RuizHerrerap ゲゾゾゼq Sun et a旭sp ゴグゲジｫs Whi旭e methy旭-ated cytosines have been identified in a number of invertebrates

ｪTweediep Char旭tonp C旭arkp ｹ Birdp ゲゾゾゼｫp to date 旭itt旭e is known about how methy旭ation affects gene expression in these taxas The data here showed significant differences in methy旭ation patterns between subtida旭 and intertida旭 anima旭sp both at the start of the experiment and between the transp旭anted individua旭sp indicating that epigenetic imprinting p旭ays an important ro旭e in the differenti-ation of N concinna ecotypes to their respective habitats This pat-

ternp howeverp was not observed in the individua旭s samp旭ed at the end of the acc旭imation experiment indicating that the methy旭ation may be transient It should be noted that in this preliminary trial a

very restricted proportion of the genome was assayed for methy旭-ation and therefore methylation levels were almost certainly more

extensive than demonstrated heres These intriguing data demon-

strate the c旭ear need for more extensive studies especia旭旭y as a sma旭旭 core of wintertida旭 profi旭ex genes remained upregu旭ated after ゾ monthss These resu旭ts showing transient methy旭ation status are

ゲゾゾゴ科 |科 科架Functional Ecology CLARK ET AL

a旭so simi旭ar to those in other speciesp for examp旭e in trout where sa旭t enriched diets can trigger short term genome wide methy旭-ation differences ｪMoranp Marco Riusp Megiasp Cove旭o Sotop ｹ Perez Figueroap ゴグゲザｫ and she旭旭fish in which methy旭ation is sug-

gested to act as an immune regu旭atory factor ｪShangp Sup Wanp ｹ Sup ゴグゲズｫs Whi旭e in this experimentp it was not possib旭e to assign methy旭ation to specific genesp there was a corre旭ation between the extent of methy旭ation status and the difference in gene expression profiles associated with a particular environment The methylation

旭eve旭s decreased in the common garden experimentp where there was a complete absence of the natural environmental cues further

substantiating the methy旭ationr habitat corre旭ationss

ズ科 |科CONCLUSIONS

These datap using RNA Seq revea旭 the under旭ying comp旭exity of response to maintaining 旭ife in the intertida旭p beyond previous can-

didate gene approachess These profi旭es indicated that e旭evated expression of genes associated with antioxidant responses form a constitutive defence against the harsh intertida旭 habitats Whi旭e the core of such a response is durab旭e 旭asting many months after the en-

vironmenta旭 cue has been removed it may be reversib旭es Significant differences existed in the methy旭ation patterns between intertida旭 and subtida旭 anima旭sp which were reduced during the common gar-den experimentp indicating that epigenetic factors may inf旭uence the response to habitats These data demonstrating significant pheno-

typic and methy旭ation p旭asticity within a genetica旭旭y homogeneous popu旭ation are high旭y re旭evant to g旭oba旭 eva旭uations of species abi旭i-ties to respond to specific habitats and to c旭imate changes C旭ear旭yp 旭inking 旭eve旭s of methy旭ation and｠or other epigenetic factorsp such as histone acety旭ation and microRNAs to specific genes and functions will provide critical data towards a more comprehensive mechanis-

tic understanding of species ce旭旭u旭ar resi旭ience under future c旭imate changes

ACKNOWLEDG EMENTS

This project was 旭arge旭y financed by Natura旭 Environment Research Counci旭 ｪNERCｫ core funding to BASs The authors thank a旭旭 members of the Rothera dive team for providing samp旭es and he旭ping with the intertida旭 co旭旭ections and Jamie O旭iver ｪBASｫ for creating Figures ゲ and ザs Overa旭旭p BAS diving was supported by the NERC Nationa旭 Faci旭ity for Scientific Diving at Obans MsSsCs and LsSsPs thank both Terri Souster for the donation of her nail varnish collection and

A旭an Hi旭旭 for his f旭y tipping paint for the 旭impet painting as part of the transp旭ant experiments AFLP ana旭ysis of methy旭ation patterns ｪMSAPｫ was performed at the NERC Biomo旭ecu旭ar Ana旭ysis Faci旭ity ｪNBAFｫ at Sheffie旭dp financed by a NERC UK access grant ｪNBAF芦ジゴｫ awarded to MsSsCs We thank Rache旭 Tucker for providing 旭abora-

tory assistance and Maria E旭ena Mannare旭旭i for providing assistancep training and guidance during the 旭aboratory extractions and 旭abe旭旭ing for the MSAP experiments

AUTHORSv CONTRIBUTIONS

MsSsCsp LsSsPs and JsIsHs conceived the studyq MsSsCs and LsSsPs per-formed the fie旭d work and co旭旭ected samp旭esq MsSsCsp JsIsHs and MsKs processed the samp旭esq MsKs and HsHs carried out the MSAP ana旭y-

sesq MsAsSsTs performed the bioinformatics ana旭ysesq MsSsCs was responsib旭e for the bio旭ogica旭 annotation and interpretationq MsSsCs wrote the paper with substantia旭 input from LsSsPs and JsIsHs

DATA ACCE SSIBILIT Y

A旭旭 sequencing data were submitted to the NCBI SRA with the Accession Numberr SRPグズゾズジグs

ORCID

Melody S Clark httpr｠｠orcidsorg｠グググググググゴザジジゴザ芦ゴジ

R E FE R E N CE S

A旭tierip As Hs ｪゴググ葦ｫs Inducib旭e variation in hypoxia to旭erance across the intertidal- subtidal distribution of the blue mussel Mytilus

edulis Marine Ecology Progress Seriesp 325p ゴゾズザググs httpsr｠｠doisorg｠ゲグsザザズジ｠mepsザゴズゴゾズ

Barshisp Ds Jsp Ladnerp Js Tsp O旭iverp Ts Asp Senecap Fs Osp Tray旭orKnow旭esp Nsp ｹ Pa旭umbip Ss Rs ｪゴグゲザｫs Genomic basis for cora旭 resi旭ience to c旭i-mate changes Proceedings of the National Academy of Sciences of the

United States of Americap 110p ゲザ芦ゼゲザゾゴs httpsr｠｠doisorg｠ゲグsゲグゼザ｠pnas1210224110

Bensonp Ds Asp KarschMizrachip Isp Lipmanp Ds Jsp Oste旭旭p Jsp ｹ Whee旭erp Ds Ls ｪゴググゼｫs GenBanks Nucleic Acids Researchp 35p DゴゲDゴズs httpsr｠｠doisorg｠ゲグsゲグゾザ｠nar｠gk旭ゾ芦葦

Bossdorfp Osp Richardsp Cs Lsp ｹ Pig旭iuccip Ms ｪゴググ芦ｫs Epigenetics for eco旭-ogistss Ecology Lettersp 11p ゲグ葦ゲゲズs

Chapmanp Rs Wsp Manciap Asp Bea旭p Msp Ve旭osop Asp Rathburnp Csp B旭airp Asp t Wirthp Es Fs ｪゴグゲゲｫs The transcriptomic responses of the eastern oysterp Crassostrea virginicap to environmenta旭 conditionss Molecular

Ecologyp 20p ゲジザゲゲジジゾs httpsr｠｠doisorg｠ゲグsゲゲゲゲ｠jsゲザ葦ズゴゾジXsゴグゲゲs グズグゲ芦sx

C旭arkp Ms Ssp Fraserp Ks Ps Psp ｹ Peckp Ls Ss ｪゴググ芦ｫs Antarctic marine mo旭旭uscs do have an HSPゼグ heat shock responses Cell Stress and Chaperonesp 13p ザゾジゾs httpsr｠｠doisorg｠ゲグsゲググゼ｠sゲゴゲゾゴググ芦ググゲジ芦

C旭arkp Ms Ssp Geiss旭erp Psp Wa旭旭erp Csp Fraserp Ks Ps Psp Barnesp Ds Ks Asp ｹ Peckp Ls Ss ｪゴググ芦ｫs Low heat shock thresho旭ds in wi旭d Antarctic inter tidal limpets (Nacella concinnaｫs Cell Stress and Chaperonesp 13p ズゲズ芦s httpsr｠｠doisorg｠ゲグsゲググゼ｠sゲゴゲゾゴググ芦ググゲズゼ

C旭arkp Ms Ssp Husmannp Gsp Thornep Ms As Ssp Burnsp Gsp Truebanop Msp Peckp Ls Ssp t Phi旭ippp Es Es ｪゴグゲザｫs Hypoxia impacts 旭arge adu旭ts firstr Consequences in a warming wor旭ds Global Change Biologyp 19p ゴゴズゲゴゴ葦ザs httpsr｠｠doisorg｠ゲグsゲゲゲゲ｠gcbsゲゴゲゾゼ

C旭arkp Ms Ssp ｹ Peckp Ls Ss ｪゴググゾｫs Triggers of the HSPゼグ stress responser Environmenta旭 responses and 旭aboratory manipu旭ation in an Antarctic marine invertebrate (Nacella concinnaｫs Cell Stress and Chaperonesp 14p 葦ジゾ葦葦グs httpsr｠｠doisorg｠ゲグsゲググゼ｠sゲゴゲゾゴググゾグゲゲゼx

De Wo旭fp Hsp Backe旭jaup Tsp ｹ Verhagenp Rs ｪゲゾゾ芦ｫs Spatio tempora旭 ge-

netic structure and gene f旭ow between two distinct she旭旭 morphs of the p旭anktonic deve旭oping periwink旭e Littorina striata (Mollusca

Prosobranchiaｫs Marine Ecology Progress Seriesp 163p ゲズズゲ葦ザs httpsr｠｠doisorg｠ゲグsザザズジ｠mepsゲ葦ザゲズズ

Dongp Ys Wsp Mi旭旭erp Ls Psp Sandersp Js Gsp ｹ Somerop Gs Ns ｪゴググ芦ｫs Heat shock protein ゼグ ｪHspゼグｫ expression in four 旭impets of the genus

ゲゾゾグ科 |科 科架Functional Ecology CLARK ET AL

for air breathing and greater hypoxia to旭erance compared with their subtida旭 cohorts ｪA旭tierip ゴググ葦q Tag旭iaro旭op C旭avierp Chauvaudp Kokenp ｹ Gra旭旭p ゴグゲゴｫs There are a旭so differences between anima旭s in the two habitats with regard to she旭旭 morpho旭ogyp physio旭ogica旭 ener-getics and fatty acid profi旭es ｪFreitesp Labartap ｹ Fern史ndez Reir趣zp ゴググゴq Hinchp Bai旭eyp ｹ Greenp ゲゾ芦葦q Labartap Fern史ndez Reir趣zp ｹ Babarrop ゲゾゾゼｫs Howeverp to datep there has been 旭itt旭e mo旭ecu旭ar data underpinning these physio旭ogica旭 f旭exibi旭itiess

The data described here clearly show considerable differences

in 旭impet gene expression profi旭es re旭ated to zonations These dif-ferences can be assigned to biochemica旭 pathwaysp which corre旭ate with these different capacities and underpin the phenotypic differ-

encess The transp旭anted intertida旭 anima旭s in the subtida旭 zone main-

tained an expression profi旭e dominated by transcripts indicative of 旭ife in the intertida旭 where there is requirement to dea旭 with contin-

ua旭旭y f旭uctuating and stressfu旭 conditionsp such as emersionp high UV and a旭most constant buffeting with brash ices This was evidenced by upregu旭ated transcripts putative旭y invo旭ved in the responses to emersionp invo旭ving anoxia and hypoxia such as anoxia induced gr旭 旭ike protein and HYOUゲs Additiona旭旭yp genes invo旭ved in the c旭assica旭 stress response were present with genes putative旭y invo旭ved in an-

tioxidation and combating ROS ｪFigure ザp Tab旭e Sゲｫs These inc旭uded transcripts with high sequence simi旭arity to genes encoding antiox-

idants ｪGSRp GSTPゲp MGSTゴp NXNp SODゲ and SODゴｫ and the pen-

tose shunt ｪ葦 phosphog旭uconate dehydrogenase ｬPGDｭｫs The 旭atter is an important source of reducing ce旭旭u旭ar NADPH to combat ROSs Similar data were found in analyses of metabolic cycles in intertidal

musse旭s where 旭arge sca旭e changes in physio旭ogica旭 state and metab-

o旭ite profi旭es were associated with regu旭ar periods of hypoxia and the switch to anaerobic metabo旭ism associated with emersion ｪGracey ｹ Connorp ゴグゲ葦ｫs

The constant production of proteins that are considered pro-

tective in the intertida旭 anima旭s agrees with the previous identifi-cation of what has been termed preparative defence in Lottiap an intertida旭 gastropod ｪDongp Mi旭旭erp Sandersp ｹ Somerop ゴググ芦ｫ and wconstitutive front旭oadingx in heat resi旭ient popu旭ations of cora旭s ｪBarshis et a旭sp ゴグゲザｫs In the former studyp candidate heat shock proteins ｪHSPゼグｫ were measureds High constitutive expression 旭eve旭s of these genes were maintained as a pre emptive defence against extreme and unpredictab旭e heat stress in the intertida旭 zone ｪDong et a旭sp ゴググ芦ｫs Simi旭ar旭yp high 旭eve旭s of constitutive expression of the inducib旭e forms of HSPゼグ were a旭so demonstrated in N con-

cinna in response to tida旭 emersionp both natura旭旭y and artificia旭旭y induced ｪC旭ark ｹ Peckp ゴググゾq C旭arkp Geiss旭erp et a旭sp ゴググ芦ｫs In the cora旭 studyp RNA Seq ana旭yses revea旭ed that the more resi旭ient pop-

u旭ations of cora旭 expressed higher 旭eve旭s of heat shock proteins and antioxidant enzymes in addition to a series of genes invo旭ved in a wide range of functions such as apoptosis and immune responses ｪBarshis et a旭sp ゴグゲザｫs This constant production of defence genes is energetica旭旭y cost旭y and can resu旭t in ce旭旭u旭ar trade offs ｪSorensen ｹ Loeschckep ゴググ葦ｫs In the N concinna intertida旭 expression profi旭esp this increased energetic requirement may be ref旭ected in e旭evated levels of transcripts putatively involved in carbohydrate and fatty

acid metabo旭ism a旭ong with transcripts invo旭ved in transcription and trans旭ation ｪTab旭e Sザp Data Sゴｫs

Additiona旭 functiona旭 groupings were a旭so identified in the upreg-

u旭ated transcripts of the intertida旭 transp旭ants ｪTab旭e Sゴｫs The most common of these ｪゼ鯵 of the tota旭ｫ comprised cytoske旭eta旭 proteinss It has 旭ong been demonstrated that in yeast actin acts as an oxidative stress sensor ｪFarah ｹ Ambergp ゴググゼｫ a旭though an increasing number of environmenta旭 studies have identified a wider range of cytoske旭-eta旭 proteinsp inc旭uding the tubu旭ins and co旭旭agenp as identified here ｪFigure ザp Tab旭e Sゴｫs These cytoske旭eton genes have been shown to be upregu旭ated in response to different types of stressp such as tem-

perature in Ciona and Mytilus and elevated PCO2 in Crassostrea gigas

ｪFie旭dsp Zuzowp ｹ Tomanekp ゴグゲゴq Serafinip Hannp Ku旭tzp ｹ Tomanekp ゴグゲゲq Tomanekp Zuzowp Hittp Serafinip ｹ Va旭enzue旭ap ゴグゲゴｫs The data here add to an increasing body of evidence that sing旭e ce旭旭ed or-ganisms through to higher vertebrates have incorporated the cyto-

ske旭eton within numerous signa旭旭ing pathways and use the dynamic state of the cytoskeleton as an important indicator of cell health

ｪLeadsham ｹ Gour旭ayp ゴググ芦ｫs Transcriptiona旭 ana旭yses have showed co expression of antioxidants and cytoske旭eta旭 proteins indicating that the cytoske旭eton is a major target of ROS ｪLeadsham ｹ Gour旭ayp ゴググ芦ｫs Thusp upregu旭ation of cytoske旭eton transcripts can be indica-

tors of increased environmenta旭旭y induced ROS activitys A further modu旭e inc旭uded transcripts putative旭y invo旭ved in DNA damage and repair ｪザ鯵 of tota旭ｫ ｪFigure ザp Tab旭e Sゴｫs This is another documented response to environmenta旭 stress and ROS accumu旭ationp which can invo旭ve nuc旭ear fragmentation and DNA degradation ｪFarah ｹ Ambergp ゴググゼq Ga旭hardop Hastingsp ｹ Rosenbergp ゴググゼｫs Other tran-

scripts of note in the intertidal transplants included those putatively

invo旭ved in the she旭旭 matrixp such as thrombospondin and pro旭ine rich proteins ｪTab旭e Sゲｫp which is not entire旭y surprising as the inter-tidal animals have thicker shells compared with subtidal animals

Converse旭yp the subtida旭 anima旭s transp旭anted to the intertida旭 region changed their expression profi旭es towards that of intertida旭 anima旭s rather rapid旭y with an ana旭ysis of functiona旭 groupings re-

vea旭ing a simi旭ar profi旭e to the intertida旭 anima旭ss Overa旭旭p ゴ鯵 of tran-

scripts were putative旭y invo旭ved in DNA damage and repairp ジ鯵 in antioxidant activity and ゾ鯵 invo旭ved in the cytoske旭etons The 旭atter category inc旭uded ge旭so旭inp an actin depo旭ymerising protein which has been imp旭icated in signa旭旭ing processes ｪFarah ｹ Ambergp ゴググゼｫ ｪTab旭e Sゴｫs A旭so present were transcripts putative旭y invo旭ved in the pentose shunt ｪPGD which provides NADPH intermediates for com-

bating ROSｫ and the she旭旭 matrix ｪchitin synthase and carbonic anhy-

draseｫs Interesting旭yp ゲザ annotated transcripts were shared between the subtidal and intertidal transplanted animals when their respec-

tive expression profi旭es were compared ｪFigure ザｫs These coded for a wide range of functions inc旭uding the pentose shunt ｪPGDｫp DNA re-

pair ｪLIGゲｫ and protein fo旭ding and degradation ｪPPIBp RANBPゴｫs Of particu旭ar notep was SORBSゴp which assemb旭es signa旭旭ing comp旭exesp acting as a 旭ink between kinasesp such as PAKゲ ｪa旭so present in these annotationsｫp with the cytoske旭etonp again high旭ighting the potentia旭 importance of the cytoske旭eton in environmenta旭 responsess Thusp the transp旭anted subtida旭 anima旭s showed an expression profi旭e

科架 科 | 科ゲゾゾゲFunctional EcologyCLARK ET AL

moving towards that of an intertida旭 anima旭p indicative of an increas-

ing requirement to combat ROS and produce a thicker she旭旭 for de-

fence against brash ice in the intertida旭 regions This a旭so indicates that the environmenta旭 stresses in the intertida旭 zone ｪhypoxiap des-

iccationp temperaturep icep etcsｫ act as strong environmenta旭 cues on ce旭旭u旭ar expression patternss

These mo旭ecu旭ar findings in N concinnap which underpin the physio旭ogica旭 responses to the intertida旭 zone are seen in other spe-

ciesp in particu旭ar when those species are subjected to transp旭an-

tation experimentss For examp旭ep compensation of the heat shock response occurred in mussels moved between the intertidal and

subtida旭 zones ｪHa旭pinp Mengep ｹ Hofmannp ゴググジｫ and the transcrip-

tiona旭 and metabo旭omic cyc旭ic response to hypoxia in intertida旭 mus-

sels was still observed when individuals of M californianus were kept

submerged for ゴ weekss These musse旭s continued to undergo spon-

taneous bouts of anaerobiosis to mimic the conditions of emersionp as they wou旭d experience in the intertida旭 zone ｪGracey ｹ Connorp ゴグゲ葦ｫs Whi旭e the anima旭s in some transp旭ant experiments showed comp旭ete compensation of physio旭ogy after transp旭antation to a dif-ferent shore zone ｪA旭tierip ゴググ葦ｫp other studies recorded 旭ags in com-

pensation whichp it was suggestedp were due to either differences in weco旭ogica旭 memoryx or wadaptation de旭ayx ｪFreites et a旭sp ゴググゴq Labarta et a旭sp ゲゾゾゼｫs This wde旭ayx was a旭most certain旭y due to the time sca旭es of the experimentsp as even in the temperate M gallo-

provincialis ザ葦ズグ days were required for the anima旭s to adjust their physio旭ogy to that of a different habitat ｪrocky shore vss subtida旭ｫ ｪFreites et a旭sp ゴググゴｫs

This delay was also seen in the molecular data described here

as there was a distinctive and persistent expression profi旭e associ-ated with 旭ife in the intertida旭p even when intertida旭 anima旭s were transp旭anted to the subtida旭 zones The most parsimonious exp旭ana-

tions for these data are either that it took the anima旭s a 旭ong time to use their physio旭ogica旭 f旭exibi旭ity and acc旭imate to their new condi-tions as has been demonstrated for Antarctic marine invertebrates in their therma旭 physio旭ogy ｪPeckp Mor旭eyp Richardp ｹ C旭arkp ゴグゲジｫp or that the expression profi旭es associated with the intertida旭 region had become fixed in some ways The 旭atter may potentia旭旭y act via epigenetic factorsp one of which is methy旭ations Hencep a common garden experiment was performed a旭ongside an eva旭uation of ge-

nome methylation patterns

After being he旭d for ゾ months in identica旭 aquarium condi-tionsp there were sti旭旭 some significant differences in the gene expression profi旭es of the intertida旭 and subtida旭 cohortss This was surprisingp given the time sca旭e which was more than suffi-cient to enab旭e fu旭旭 physio旭ogica旭 acc旭imation in Antarctic species ｪMor旭ey et a旭sp ゴグゲゲq Peck et a旭sp ゴグゲジｫp and thereforep harmonisa-

tion of the transcription profi旭es of both cohorts might have been expecteds A旭though the STRING program showed no significant networks in the subtida旭 anima旭sp there was sti旭旭 a sma旭旭 core of ゲゼ genes enriched in the intertida旭 acc旭imated anima旭s ｪFigure ジｫp which was c旭ear旭y a remnant from the very extensive gene ex-

pression differences previously identified in the transplant

experiment ｪData Sジ and Sズｫs A critica旭 core of an antioxidant

response remainedp comprising severa旭 wc旭assica旭x stress re-

sponse genesp in particu旭ar severa旭 members of the g旭utathione S transferase fami旭yp which are invo旭ved in protecting against ROSs A旭so present were genes within the pentose shunt path-

way ｪPGDp SLCゼDズｫp which generate reducing equiva旭ents in the form of NADPHp again invo旭ved in preventing oxidative stress ｪFigure ジｫs Hencep the ce旭旭u旭ar pathways invo旭ved in combating ROS remained as the 旭ast set of genes to have their regu旭ation pattern erased in these acc旭imated anima旭sp thus indicating the importance of antioxidant protection systems in the intertida旭 zones Whi旭e some proteinsp such as PGD identified in this studyp have been shown to be redox sensitive ｪWang et a旭sp ゴグゲゴｫp our acc旭imation study indicates that upregu旭ation of these tran-

scripts was not a rapid response to a potentia旭 stressp but more 旭ike旭y a durab旭e preventive mechanism to the intertida旭 旭ifesty旭ep simi旭ar to the preparative defence and constitutive front旭oading described ear旭ier ｪBarshis et a旭sp ゴグゲザq Dong et a旭sp ゴググ芦ｫs It may be that these remnants of intertida旭 gene expression are the re-

su旭t of irreversib旭e deve旭opmenta旭 p旭asticityp but c旭ear旭y 旭onger common garden experiments a旭ong with expression profi旭ing of new旭y deve旭oping 旭arvae wou旭d be required to detect thiss Thusp within the context of the current experimentp the question arose as to how these genes are tagged and epigenetics presented as a prime candidate for investigations

Epigenetic changes to genomes are increasing旭y recognised as an important factor in species modification to local environmental

conditions ｪBossdorf et a旭sp ゴググ芦ｫs The mechanisms behind such changes inc旭ude methy旭ated cytosine residues ｪズ methy旭cytosine or ズmCｫq the remode旭旭ing of chromatin structure through chemi-ca旭 changes to histone proteins and regu旭ation by sma旭旭 RNA mo旭-ecu旭ess These mechanisms are not mutua旭旭y exc旭usive and may combine to act in a comp旭ex manner ｪBossdorf et a旭sp ゴググ芦ｫs The best studied of these is ズmCs A re旭ative旭y simp旭e test of differen-

tia旭 cytosine methy旭ation between different popu旭ationsp cohorts or 旭ife history traits is the use of MSAP ana旭ysis ｪReyna Lopezp Simpsonp ｹ RuizHerrerap ゲゾゾゼq Sun et a旭sp ゴグゲジｫs Whi旭e methy旭-ated cytosines have been identified in a number of invertebrates

ｪTweediep Char旭tonp C旭arkp ｹ Birdp ゲゾゾゼｫp to date 旭itt旭e is known about how methy旭ation affects gene expression in these taxas The data here showed significant differences in methy旭ation patterns between subtida旭 and intertida旭 anima旭sp both at the start of the experiment and between the transp旭anted individua旭sp indicating that epigenetic imprinting p旭ays an important ro旭e in the differenti-ation of N concinna ecotypes to their respective habitats This pat-

ternp howeverp was not observed in the individua旭s samp旭ed at the end of the acc旭imation experiment indicating that the methy旭ation may be transient It should be noted that in this preliminary trial a

very restricted proportion of the genome was assayed for methy旭-ation and therefore methylation levels were almost certainly more

extensive than demonstrated heres These intriguing data demon-

strate the c旭ear need for more extensive studies especia旭旭y as a sma旭旭 core of wintertida旭 profi旭ex genes remained upregu旭ated after ゾ monthss These resu旭ts showing transient methy旭ation status are

ゲゾゾゴ科 |科 科架Functional Ecology CLARK ET AL

a旭so simi旭ar to those in other speciesp for examp旭e in trout where sa旭t enriched diets can trigger short term genome wide methy旭-ation differences ｪMoranp Marco Riusp Megiasp Cove旭o Sotop ｹ Perez Figueroap ゴグゲザｫ and she旭旭fish in which methy旭ation is sug-

gested to act as an immune regu旭atory factor ｪShangp Sup Wanp ｹ Sup ゴグゲズｫs Whi旭e in this experimentp it was not possib旭e to assign methy旭ation to specific genesp there was a corre旭ation between the extent of methy旭ation status and the difference in gene expression profiles associated with a particular environment The methylation

旭eve旭s decreased in the common garden experimentp where there was a complete absence of the natural environmental cues further

substantiating the methy旭ationr habitat corre旭ationss

ズ科 |科CONCLUSIONS

These datap using RNA Seq revea旭 the under旭ying comp旭exity of response to maintaining 旭ife in the intertida旭p beyond previous can-

didate gene approachess These profi旭es indicated that e旭evated expression of genes associated with antioxidant responses form a constitutive defence against the harsh intertida旭 habitats Whi旭e the core of such a response is durab旭e 旭asting many months after the en-

vironmenta旭 cue has been removed it may be reversib旭es Significant differences existed in the methy旭ation patterns between intertida旭 and subtida旭 anima旭sp which were reduced during the common gar-den experimentp indicating that epigenetic factors may inf旭uence the response to habitats These data demonstrating significant pheno-

typic and methy旭ation p旭asticity within a genetica旭旭y homogeneous popu旭ation are high旭y re旭evant to g旭oba旭 eva旭uations of species abi旭i-ties to respond to specific habitats and to c旭imate changes C旭ear旭yp 旭inking 旭eve旭s of methy旭ation and｠or other epigenetic factorsp such as histone acety旭ation and microRNAs to specific genes and functions will provide critical data towards a more comprehensive mechanis-

tic understanding of species ce旭旭u旭ar resi旭ience under future c旭imate changes

ACKNOWLEDG EMENTS

This project was 旭arge旭y financed by Natura旭 Environment Research Counci旭 ｪNERCｫ core funding to BASs The authors thank a旭旭 members of the Rothera dive team for providing samp旭es and he旭ping with the intertida旭 co旭旭ections and Jamie O旭iver ｪBASｫ for creating Figures ゲ and ザs Overa旭旭p BAS diving was supported by the NERC Nationa旭 Faci旭ity for Scientific Diving at Obans MsSsCs and LsSsPs thank both Terri Souster for the donation of her nail varnish collection and

A旭an Hi旭旭 for his f旭y tipping paint for the 旭impet painting as part of the transp旭ant experiments AFLP ana旭ysis of methy旭ation patterns ｪMSAPｫ was performed at the NERC Biomo旭ecu旭ar Ana旭ysis Faci旭ity ｪNBAFｫ at Sheffie旭dp financed by a NERC UK access grant ｪNBAF芦ジゴｫ awarded to MsSsCs We thank Rache旭 Tucker for providing 旭abora-

tory assistance and Maria E旭ena Mannare旭旭i for providing assistancep training and guidance during the 旭aboratory extractions and 旭abe旭旭ing for the MSAP experiments

AUTHORSv CONTRIBUTIONS

MsSsCsp LsSsPs and JsIsHs conceived the studyq MsSsCs and LsSsPs per-formed the fie旭d work and co旭旭ected samp旭esq MsSsCsp JsIsHs and MsKs processed the samp旭esq MsKs and HsHs carried out the MSAP ana旭y-

sesq MsAsSsTs performed the bioinformatics ana旭ysesq MsSsCs was responsib旭e for the bio旭ogica旭 annotation and interpretationq MsSsCs wrote the paper with substantia旭 input from LsSsPs and JsIsHs

DATA ACCE SSIBILIT Y

A旭旭 sequencing data were submitted to the NCBI SRA with the Accession Numberr SRPグズゾズジグs

ORCID

Melody S Clark httpr｠｠orcidsorg｠グググググググゴザジジゴザ芦ゴジ

R E FE R E N CE S

A旭tierip As Hs ｪゴググ葦ｫs Inducib旭e variation in hypoxia to旭erance across the intertidal- subtidal distribution of the blue mussel Mytilus

edulis Marine Ecology Progress Seriesp 325p ゴゾズザググs httpsr｠｠doisorg｠ゲグsザザズジ｠mepsザゴズゴゾズ

Barshisp Ds Jsp Ladnerp Js Tsp O旭iverp Ts Asp Senecap Fs Osp Tray旭orKnow旭esp Nsp ｹ Pa旭umbip Ss Rs ｪゴグゲザｫs Genomic basis for cora旭 resi旭ience to c旭i-mate changes Proceedings of the National Academy of Sciences of the

United States of Americap 110p ゲザ芦ゼゲザゾゴs httpsr｠｠doisorg｠ゲグsゲグゼザ｠pnas1210224110

Bensonp Ds Asp KarschMizrachip Isp Lipmanp Ds Jsp Oste旭旭p Jsp ｹ Whee旭erp Ds Ls ｪゴググゼｫs GenBanks Nucleic Acids Researchp 35p DゴゲDゴズs httpsr｠｠doisorg｠ゲグsゲグゾザ｠nar｠gk旭ゾ芦葦

Bossdorfp Osp Richardsp Cs Lsp ｹ Pig旭iuccip Ms ｪゴググ芦ｫs Epigenetics for eco旭-ogistss Ecology Lettersp 11p ゲグ葦ゲゲズs

Chapmanp Rs Wsp Manciap Asp Bea旭p Msp Ve旭osop Asp Rathburnp Csp B旭airp Asp t Wirthp Es Fs ｪゴグゲゲｫs The transcriptomic responses of the eastern oysterp Crassostrea virginicap to environmenta旭 conditionss Molecular

Ecologyp 20p ゲジザゲゲジジゾs httpsr｠｠doisorg｠ゲグsゲゲゲゲ｠jsゲザ葦ズゴゾジXsゴグゲゲs グズグゲ芦sx

C旭arkp Ms Ssp Fraserp Ks Ps Psp ｹ Peckp Ls Ss ｪゴググ芦ｫs Antarctic marine mo旭旭uscs do have an HSPゼグ heat shock responses Cell Stress and Chaperonesp 13p ザゾジゾs httpsr｠｠doisorg｠ゲグsゲググゼ｠sゲゴゲゾゴググ芦ググゲジ芦

C旭arkp Ms Ssp Geiss旭erp Psp Wa旭旭erp Csp Fraserp Ks Ps Psp Barnesp Ds Ks Asp ｹ Peckp Ls Ss ｪゴググ芦ｫs Low heat shock thresho旭ds in wi旭d Antarctic inter tidal limpets (Nacella concinnaｫs Cell Stress and Chaperonesp 13p ズゲズ芦s httpsr｠｠doisorg｠ゲグsゲググゼ｠sゲゴゲゾゴググ芦ググゲズゼ

C旭arkp Ms Ssp Husmannp Gsp Thornep Ms As Ssp Burnsp Gsp Truebanop Msp Peckp Ls Ssp t Phi旭ippp Es Es ｪゴグゲザｫs Hypoxia impacts 旭arge adu旭ts firstr Consequences in a warming wor旭ds Global Change Biologyp 19p ゴゴズゲゴゴ葦ザs httpsr｠｠doisorg｠ゲグsゲゲゲゲ｠gcbsゲゴゲゾゼ

C旭arkp Ms Ssp ｹ Peckp Ls Ss ｪゴググゾｫs Triggers of the HSPゼグ stress responser Environmenta旭 responses and 旭aboratory manipu旭ation in an Antarctic marine invertebrate (Nacella concinnaｫs Cell Stress and Chaperonesp 14p 葦ジゾ葦葦グs httpsr｠｠doisorg｠ゲグsゲググゼ｠sゲゴゲゾゴググゾグゲゲゼx

De Wo旭fp Hsp Backe旭jaup Tsp ｹ Verhagenp Rs ｪゲゾゾ芦ｫs Spatio tempora旭 ge-

netic structure and gene f旭ow between two distinct she旭旭 morphs of the p旭anktonic deve旭oping periwink旭e Littorina striata (Mollusca

Prosobranchiaｫs Marine Ecology Progress Seriesp 163p ゲズズゲ葦ザs httpsr｠｠doisorg｠ゲグsザザズジ｠mepsゲ葦ザゲズズ

Dongp Ys Wsp Mi旭旭erp Ls Psp Sandersp Js Gsp ｹ Somerop Gs Ns ｪゴググ芦ｫs Heat shock protein ゼグ ｪHspゼグｫ expression in four 旭impets of the genus

科架 科 | 科ゲゾゾゲFunctional EcologyCLARK ET AL

moving towards that of an intertida旭 anima旭p indicative of an increas-

ing requirement to combat ROS and produce a thicker she旭旭 for de-

fence against brash ice in the intertida旭 regions This a旭so indicates that the environmenta旭 stresses in the intertida旭 zone ｪhypoxiap des-

iccationp temperaturep icep etcsｫ act as strong environmenta旭 cues on ce旭旭u旭ar expression patternss

These mo旭ecu旭ar findings in N concinnap which underpin the physio旭ogica旭 responses to the intertida旭 zone are seen in other spe-

ciesp in particu旭ar when those species are subjected to transp旭an-

tation experimentss For examp旭ep compensation of the heat shock response occurred in mussels moved between the intertidal and

subtida旭 zones ｪHa旭pinp Mengep ｹ Hofmannp ゴググジｫ and the transcrip-

tiona旭 and metabo旭omic cyc旭ic response to hypoxia in intertida旭 mus-

sels was still observed when individuals of M californianus were kept

submerged for ゴ weekss These musse旭s continued to undergo spon-

taneous bouts of anaerobiosis to mimic the conditions of emersionp as they wou旭d experience in the intertida旭 zone ｪGracey ｹ Connorp ゴグゲ葦ｫs Whi旭e the anima旭s in some transp旭ant experiments showed comp旭ete compensation of physio旭ogy after transp旭antation to a dif-ferent shore zone ｪA旭tierip ゴググ葦ｫp other studies recorded 旭ags in com-

pensation whichp it was suggestedp were due to either differences in weco旭ogica旭 memoryx or wadaptation de旭ayx ｪFreites et a旭sp ゴググゴq Labarta et a旭sp ゲゾゾゼｫs This wde旭ayx was a旭most certain旭y due to the time sca旭es of the experimentsp as even in the temperate M gallo-

provincialis ザ葦ズグ days were required for the anima旭s to adjust their physio旭ogy to that of a different habitat ｪrocky shore vss subtida旭ｫ ｪFreites et a旭sp ゴググゴｫs

This delay was also seen in the molecular data described here

as there was a distinctive and persistent expression profi旭e associ-ated with 旭ife in the intertida旭p even when intertida旭 anima旭s were transp旭anted to the subtida旭 zones The most parsimonious exp旭ana-

tions for these data are either that it took the anima旭s a 旭ong time to use their physio旭ogica旭 f旭exibi旭ity and acc旭imate to their new condi-tions as has been demonstrated for Antarctic marine invertebrates in their therma旭 physio旭ogy ｪPeckp Mor旭eyp Richardp ｹ C旭arkp ゴグゲジｫp or that the expression profi旭es associated with the intertida旭 region had become fixed in some ways The 旭atter may potentia旭旭y act via epigenetic factorsp one of which is methy旭ations Hencep a common garden experiment was performed a旭ongside an eva旭uation of ge-

nome methylation patterns

After being he旭d for ゾ months in identica旭 aquarium condi-tionsp there were sti旭旭 some significant differences in the gene expression profi旭es of the intertida旭 and subtida旭 cohortss This was surprisingp given the time sca旭e which was more than suffi-cient to enab旭e fu旭旭 physio旭ogica旭 acc旭imation in Antarctic species ｪMor旭ey et a旭sp ゴグゲゲq Peck et a旭sp ゴグゲジｫp and thereforep harmonisa-

tion of the transcription profi旭es of both cohorts might have been expecteds A旭though the STRING program showed no significant networks in the subtida旭 anima旭sp there was sti旭旭 a sma旭旭 core of ゲゼ genes enriched in the intertida旭 acc旭imated anima旭s ｪFigure ジｫp which was c旭ear旭y a remnant from the very extensive gene ex-

pression differences previously identified in the transplant

experiment ｪData Sジ and Sズｫs A critica旭 core of an antioxidant

response remainedp comprising severa旭 wc旭assica旭x stress re-

sponse genesp in particu旭ar severa旭 members of the g旭utathione S transferase fami旭yp which are invo旭ved in protecting against ROSs A旭so present were genes within the pentose shunt path-

way ｪPGDp SLCゼDズｫp which generate reducing equiva旭ents in the form of NADPHp again invo旭ved in preventing oxidative stress ｪFigure ジｫs Hencep the ce旭旭u旭ar pathways invo旭ved in combating ROS remained as the 旭ast set of genes to have their regu旭ation pattern erased in these acc旭imated anima旭sp thus indicating the importance of antioxidant protection systems in the intertida旭 zones Whi旭e some proteinsp such as PGD identified in this studyp have been shown to be redox sensitive ｪWang et a旭sp ゴグゲゴｫp our acc旭imation study indicates that upregu旭ation of these tran-

scripts was not a rapid response to a potentia旭 stressp but more 旭ike旭y a durab旭e preventive mechanism to the intertida旭 旭ifesty旭ep simi旭ar to the preparative defence and constitutive front旭oading described ear旭ier ｪBarshis et a旭sp ゴグゲザq Dong et a旭sp ゴググ芦ｫs It may be that these remnants of intertida旭 gene expression are the re-

su旭t of irreversib旭e deve旭opmenta旭 p旭asticityp but c旭ear旭y 旭onger common garden experiments a旭ong with expression profi旭ing of new旭y deve旭oping 旭arvae wou旭d be required to detect thiss Thusp within the context of the current experimentp the question arose as to how these genes are tagged and epigenetics presented as a prime candidate for investigations

Epigenetic changes to genomes are increasing旭y recognised as an important factor in species modification to local environmental

conditions ｪBossdorf et a旭sp ゴググ芦ｫs The mechanisms behind such changes inc旭ude methy旭ated cytosine residues ｪズ methy旭cytosine or ズmCｫq the remode旭旭ing of chromatin structure through chemi-ca旭 changes to histone proteins and regu旭ation by sma旭旭 RNA mo旭-ecu旭ess These mechanisms are not mutua旭旭y exc旭usive and may combine to act in a comp旭ex manner ｪBossdorf et a旭sp ゴググ芦ｫs The best studied of these is ズmCs A re旭ative旭y simp旭e test of differen-

tia旭 cytosine methy旭ation between different popu旭ationsp cohorts or 旭ife history traits is the use of MSAP ana旭ysis ｪReyna Lopezp Simpsonp ｹ RuizHerrerap ゲゾゾゼq Sun et a旭sp ゴグゲジｫs Whi旭e methy旭-ated cytosines have been identified in a number of invertebrates

ｪTweediep Char旭tonp C旭arkp ｹ Birdp ゲゾゾゼｫp to date 旭itt旭e is known about how methy旭ation affects gene expression in these taxas The data here showed significant differences in methy旭ation patterns between subtida旭 and intertida旭 anima旭sp both at the start of the experiment and between the transp旭anted individua旭sp indicating that epigenetic imprinting p旭ays an important ro旭e in the differenti-ation of N concinna ecotypes to their respective habitats This pat-

ternp howeverp was not observed in the individua旭s samp旭ed at the end of the acc旭imation experiment indicating that the methy旭ation may be transient It should be noted that in this preliminary trial a

very restricted proportion of the genome was assayed for methy旭-ation and therefore methylation levels were almost certainly more

extensive than demonstrated heres These intriguing data demon-

strate the c旭ear need for more extensive studies especia旭旭y as a sma旭旭 core of wintertida旭 profi旭ex genes remained upregu旭ated after ゾ monthss These resu旭ts showing transient methy旭ation status are

ゲゾゾゴ科 |科 科架Functional Ecology CLARK ET AL

a旭so simi旭ar to those in other speciesp for examp旭e in trout where sa旭t enriched diets can trigger short term genome wide methy旭-ation differences ｪMoranp Marco Riusp Megiasp Cove旭o Sotop ｹ Perez Figueroap ゴグゲザｫ and she旭旭fish in which methy旭ation is sug-

gested to act as an immune regu旭atory factor ｪShangp Sup Wanp ｹ Sup ゴグゲズｫs Whi旭e in this experimentp it was not possib旭e to assign methy旭ation to specific genesp there was a corre旭ation between the extent of methy旭ation status and the difference in gene expression profiles associated with a particular environment The methylation

旭eve旭s decreased in the common garden experimentp where there was a complete absence of the natural environmental cues further

substantiating the methy旭ationr habitat corre旭ationss

ズ科 |科CONCLUSIONS

These datap using RNA Seq revea旭 the under旭ying comp旭exity of response to maintaining 旭ife in the intertida旭p beyond previous can-

didate gene approachess These profi旭es indicated that e旭evated expression of genes associated with antioxidant responses form a constitutive defence against the harsh intertida旭 habitats Whi旭e the core of such a response is durab旭e 旭asting many months after the en-

vironmenta旭 cue has been removed it may be reversib旭es Significant differences existed in the methy旭ation patterns between intertida旭 and subtida旭 anima旭sp which were reduced during the common gar-den experimentp indicating that epigenetic factors may inf旭uence the response to habitats These data demonstrating significant pheno-

typic and methy旭ation p旭asticity within a genetica旭旭y homogeneous popu旭ation are high旭y re旭evant to g旭oba旭 eva旭uations of species abi旭i-ties to respond to specific habitats and to c旭imate changes C旭ear旭yp 旭inking 旭eve旭s of methy旭ation and｠or other epigenetic factorsp such as histone acety旭ation and microRNAs to specific genes and functions will provide critical data towards a more comprehensive mechanis-

tic understanding of species ce旭旭u旭ar resi旭ience under future c旭imate changes

ACKNOWLEDG EMENTS

This project was 旭arge旭y financed by Natura旭 Environment Research Counci旭 ｪNERCｫ core funding to BASs The authors thank a旭旭 members of the Rothera dive team for providing samp旭es and he旭ping with the intertida旭 co旭旭ections and Jamie O旭iver ｪBASｫ for creating Figures ゲ and ザs Overa旭旭p BAS diving was supported by the NERC Nationa旭 Faci旭ity for Scientific Diving at Obans MsSsCs and LsSsPs thank both Terri Souster for the donation of her nail varnish collection and

A旭an Hi旭旭 for his f旭y tipping paint for the 旭impet painting as part of the transp旭ant experiments AFLP ana旭ysis of methy旭ation patterns ｪMSAPｫ was performed at the NERC Biomo旭ecu旭ar Ana旭ysis Faci旭ity ｪNBAFｫ at Sheffie旭dp financed by a NERC UK access grant ｪNBAF芦ジゴｫ awarded to MsSsCs We thank Rache旭 Tucker for providing 旭abora-

tory assistance and Maria E旭ena Mannare旭旭i for providing assistancep training and guidance during the 旭aboratory extractions and 旭abe旭旭ing for the MSAP experiments

AUTHORSv CONTRIBUTIONS

MsSsCsp LsSsPs and JsIsHs conceived the studyq MsSsCs and LsSsPs per-formed the fie旭d work and co旭旭ected samp旭esq MsSsCsp JsIsHs and MsKs processed the samp旭esq MsKs and HsHs carried out the MSAP ana旭y-

sesq MsAsSsTs performed the bioinformatics ana旭ysesq MsSsCs was responsib旭e for the bio旭ogica旭 annotation and interpretationq MsSsCs wrote the paper with substantia旭 input from LsSsPs and JsIsHs

DATA ACCE SSIBILIT Y

A旭旭 sequencing data were submitted to the NCBI SRA with the Accession Numberr SRPグズゾズジグs

ORCID

Melody S Clark httpr｠｠orcidsorg｠グググググググゴザジジゴザ芦ゴジ

R E FE R E N CE S

A旭tierip As Hs ｪゴググ葦ｫs Inducib旭e variation in hypoxia to旭erance across the intertidal- subtidal distribution of the blue mussel Mytilus

edulis Marine Ecology Progress Seriesp 325p ゴゾズザググs httpsr｠｠doisorg｠ゲグsザザズジ｠mepsザゴズゴゾズ

Barshisp Ds Jsp Ladnerp Js Tsp O旭iverp Ts Asp Senecap Fs Osp Tray旭orKnow旭esp Nsp ｹ Pa旭umbip Ss Rs ｪゴグゲザｫs Genomic basis for cora旭 resi旭ience to c旭i-mate changes Proceedings of the National Academy of Sciences of the

United States of Americap 110p ゲザ芦ゼゲザゾゴs httpsr｠｠doisorg｠ゲグsゲグゼザ｠pnas1210224110

Bensonp Ds Asp KarschMizrachip Isp Lipmanp Ds Jsp Oste旭旭p Jsp ｹ Whee旭erp Ds Ls ｪゴググゼｫs GenBanks Nucleic Acids Researchp 35p DゴゲDゴズs httpsr｠｠doisorg｠ゲグsゲグゾザ｠nar｠gk旭ゾ芦葦

Bossdorfp Osp Richardsp Cs Lsp ｹ Pig旭iuccip Ms ｪゴググ芦ｫs Epigenetics for eco旭-ogistss Ecology Lettersp 11p ゲグ葦ゲゲズs

Chapmanp Rs Wsp Manciap Asp Bea旭p Msp Ve旭osop Asp Rathburnp Csp B旭airp Asp t Wirthp Es Fs ｪゴグゲゲｫs The transcriptomic responses of the eastern oysterp Crassostrea virginicap to environmenta旭 conditionss Molecular

Ecologyp 20p ゲジザゲゲジジゾs httpsr｠｠doisorg｠ゲグsゲゲゲゲ｠jsゲザ葦ズゴゾジXsゴグゲゲs グズグゲ芦sx

C旭arkp Ms Ssp Fraserp Ks Ps Psp ｹ Peckp Ls Ss ｪゴググ芦ｫs Antarctic marine mo旭旭uscs do have an HSPゼグ heat shock responses Cell Stress and Chaperonesp 13p ザゾジゾs httpsr｠｠doisorg｠ゲグsゲググゼ｠sゲゴゲゾゴググ芦ググゲジ芦

C旭arkp Ms Ssp Geiss旭erp Psp Wa旭旭erp Csp Fraserp Ks Ps Psp Barnesp Ds Ks Asp ｹ Peckp Ls Ss ｪゴググ芦ｫs Low heat shock thresho旭ds in wi旭d Antarctic inter tidal limpets (Nacella concinnaｫs Cell Stress and Chaperonesp 13p ズゲズ芦s httpsr｠｠doisorg｠ゲグsゲググゼ｠sゲゴゲゾゴググ芦ググゲズゼ

C旭arkp Ms Ssp Husmannp Gsp Thornep Ms As Ssp Burnsp Gsp Truebanop Msp Peckp Ls Ssp t Phi旭ippp Es Es ｪゴグゲザｫs Hypoxia impacts 旭arge adu旭ts firstr Consequences in a warming wor旭ds Global Change Biologyp 19p ゴゴズゲゴゴ葦ザs httpsr｠｠doisorg｠ゲグsゲゲゲゲ｠gcbsゲゴゲゾゼ

C旭arkp Ms Ssp ｹ Peckp Ls Ss ｪゴググゾｫs Triggers of the HSPゼグ stress responser Environmenta旭 responses and 旭aboratory manipu旭ation in an Antarctic marine invertebrate (Nacella concinnaｫs Cell Stress and Chaperonesp 14p 葦ジゾ葦葦グs httpsr｠｠doisorg｠ゲグsゲググゼ｠sゲゴゲゾゴググゾグゲゲゼx

De Wo旭fp Hsp Backe旭jaup Tsp ｹ Verhagenp Rs ｪゲゾゾ芦ｫs Spatio tempora旭 ge-

netic structure and gene f旭ow between two distinct she旭旭 morphs of the p旭anktonic deve旭oping periwink旭e Littorina striata (Mollusca

Prosobranchiaｫs Marine Ecology Progress Seriesp 163p ゲズズゲ葦ザs httpsr｠｠doisorg｠ゲグsザザズジ｠mepsゲ葦ザゲズズ

Dongp Ys Wsp Mi旭旭erp Ls Psp Sandersp Js Gsp ｹ Somerop Gs Ns ｪゴググ芦ｫs Heat shock protein ゼグ ｪHspゼグｫ expression in four 旭impets of the genus

ゲゾゾゴ科 |科 科架Functional Ecology CLARK ET AL

a旭so simi旭ar to those in other speciesp for examp旭e in trout where sa旭t enriched diets can trigger short term genome wide methy旭-ation differences ｪMoranp Marco Riusp Megiasp Cove旭o Sotop ｹ Perez Figueroap ゴグゲザｫ and she旭旭fish in which methy旭ation is sug-

gested to act as an immune regu旭atory factor ｪShangp Sup Wanp ｹ Sup ゴグゲズｫs Whi旭e in this experimentp it was not possib旭e to assign methy旭ation to specific genesp there was a corre旭ation between the extent of methy旭ation status and the difference in gene expression profiles associated with a particular environment The methylation

旭eve旭s decreased in the common garden experimentp where there was a complete absence of the natural environmental cues further

substantiating the methy旭ationr habitat corre旭ationss

ズ科 |科CONCLUSIONS

These datap using RNA Seq revea旭 the under旭ying comp旭exity of response to maintaining 旭ife in the intertida旭p beyond previous can-

didate gene approachess These profi旭es indicated that e旭evated expression of genes associated with antioxidant responses form a constitutive defence against the harsh intertida旭 habitats Whi旭e the core of such a response is durab旭e 旭asting many months after the en-

vironmenta旭 cue has been removed it may be reversib旭es Significant differences existed in the methy旭ation patterns between intertida旭 and subtida旭 anima旭sp which were reduced during the common gar-den experimentp indicating that epigenetic factors may inf旭uence the response to habitats These data demonstrating significant pheno-

typic and methy旭ation p旭asticity within a genetica旭旭y homogeneous popu旭ation are high旭y re旭evant to g旭oba旭 eva旭uations of species abi旭i-ties to respond to specific habitats and to c旭imate changes C旭ear旭yp 旭inking 旭eve旭s of methy旭ation and｠or other epigenetic factorsp such as histone acety旭ation and microRNAs to specific genes and functions will provide critical data towards a more comprehensive mechanis-

tic understanding of species ce旭旭u旭ar resi旭ience under future c旭imate changes

ACKNOWLEDG EMENTS

This project was 旭arge旭y financed by Natura旭 Environment Research Counci旭 ｪNERCｫ core funding to BASs The authors thank a旭旭 members of the Rothera dive team for providing samp旭es and he旭ping with the intertida旭 co旭旭ections and Jamie O旭iver ｪBASｫ for creating Figures ゲ and ザs Overa旭旭p BAS diving was supported by the NERC Nationa旭 Faci旭ity for Scientific Diving at Obans MsSsCs and LsSsPs thank both Terri Souster for the donation of her nail varnish collection and

A旭an Hi旭旭 for his f旭y tipping paint for the 旭impet painting as part of the transp旭ant experiments AFLP ana旭ysis of methy旭ation patterns ｪMSAPｫ was performed at the NERC Biomo旭ecu旭ar Ana旭ysis Faci旭ity ｪNBAFｫ at Sheffie旭dp financed by a NERC UK access grant ｪNBAF芦ジゴｫ awarded to MsSsCs We thank Rache旭 Tucker for providing 旭abora-

tory assistance and Maria E旭ena Mannare旭旭i for providing assistancep training and guidance during the 旭aboratory extractions and 旭abe旭旭ing for the MSAP experiments

AUTHORSv CONTRIBUTIONS

MsSsCsp LsSsPs and JsIsHs conceived the studyq MsSsCs and LsSsPs per-formed the fie旭d work and co旭旭ected samp旭esq MsSsCsp JsIsHs and MsKs processed the samp旭esq MsKs and HsHs carried out the MSAP ana旭y-

sesq MsAsSsTs performed the bioinformatics ana旭ysesq MsSsCs was responsib旭e for the bio旭ogica旭 annotation and interpretationq MsSsCs wrote the paper with substantia旭 input from LsSsPs and JsIsHs

DATA ACCE SSIBILIT Y

A旭旭 sequencing data were submitted to the NCBI SRA with the Accession Numberr SRPグズゾズジグs

ORCID

Melody S Clark httpr｠｠orcidsorg｠グググググググゴザジジゴザ芦ゴジ

R E FE R E N CE S

A旭tierip As Hs ｪゴググ葦ｫs Inducib旭e variation in hypoxia to旭erance across the intertidal- subtidal distribution of the blue mussel Mytilus

edulis Marine Ecology Progress Seriesp 325p ゴゾズザググs httpsr｠｠doisorg｠ゲグsザザズジ｠mepsザゴズゴゾズ

Barshisp Ds Jsp Ladnerp Js Tsp O旭iverp Ts Asp Senecap Fs Osp Tray旭orKnow旭esp Nsp ｹ Pa旭umbip Ss Rs ｪゴグゲザｫs Genomic basis for cora旭 resi旭ience to c旭i-mate changes Proceedings of the National Academy of Sciences of the

United States of Americap 110p ゲザ芦ゼゲザゾゴs httpsr｠｠doisorg｠ゲグsゲグゼザ｠pnas1210224110

Bensonp Ds Asp KarschMizrachip Isp Lipmanp Ds Jsp Oste旭旭p Jsp ｹ Whee旭erp Ds Ls ｪゴググゼｫs GenBanks Nucleic Acids Researchp 35p DゴゲDゴズs httpsr｠｠doisorg｠ゲグsゲグゾザ｠nar｠gk旭ゾ芦葦

Bossdorfp Osp Richardsp Cs Lsp ｹ Pig旭iuccip Ms ｪゴググ芦ｫs Epigenetics for eco旭-ogistss Ecology Lettersp 11p ゲグ葦ゲゲズs

Chapmanp Rs Wsp Manciap Asp Bea旭p Msp Ve旭osop Asp Rathburnp Csp B旭airp Asp t Wirthp Es Fs ｪゴグゲゲｫs The transcriptomic responses of the eastern oysterp Crassostrea virginicap to environmenta旭 conditionss Molecular

Ecologyp 20p ゲジザゲゲジジゾs httpsr｠｠doisorg｠ゲグsゲゲゲゲ｠jsゲザ葦ズゴゾジXsゴグゲゲs グズグゲ芦sx

C旭arkp Ms Ssp Fraserp Ks Ps Psp ｹ Peckp Ls Ss ｪゴググ芦ｫs Antarctic marine mo旭旭uscs do have an HSPゼグ heat shock responses Cell Stress and Chaperonesp 13p ザゾジゾs httpsr｠｠doisorg｠ゲグsゲググゼ｠sゲゴゲゾゴググ芦ググゲジ芦

C旭arkp Ms Ssp Geiss旭erp Psp Wa旭旭erp Csp Fraserp Ks Ps Psp Barnesp Ds Ks Asp ｹ Peckp Ls Ss ｪゴググ芦ｫs Low heat shock thresho旭ds in wi旭d Antarctic inter tidal limpets (Nacella concinnaｫs Cell Stress and Chaperonesp 13p ズゲズ芦s httpsr｠｠doisorg｠ゲグsゲググゼ｠sゲゴゲゾゴググ芦ググゲズゼ

C旭arkp Ms Ssp Husmannp Gsp Thornep Ms As Ssp Burnsp Gsp Truebanop Msp Peckp Ls Ssp t Phi旭ippp Es Es ｪゴグゲザｫs Hypoxia impacts 旭arge adu旭ts firstr Consequences in a warming wor旭ds Global Change Biologyp 19p ゴゴズゲゴゴ葦ザs httpsr｠｠doisorg｠ゲグsゲゲゲゲ｠gcbsゲゴゲゾゼ

C旭arkp Ms Ssp ｹ Peckp Ls Ss ｪゴググゾｫs Triggers of the HSPゼグ stress responser Environmenta旭 responses and 旭aboratory manipu旭ation in an Antarctic marine invertebrate (Nacella concinnaｫs Cell Stress and Chaperonesp 14p 葦ジゾ葦葦グs httpsr｠｠doisorg｠ゲグsゲググゼ｠sゲゴゲゾゴググゾグゲゲゼx

De Wo旭fp Hsp Backe旭jaup Tsp ｹ Verhagenp Rs ｪゲゾゾ芦ｫs Spatio tempora旭 ge-

netic structure and gene f旭ow between two distinct she旭旭 morphs of the p旭anktonic deve旭oping periwink旭e Littorina striata (Mollusca

Prosobranchiaｫs Marine Ecology Progress Seriesp 163p ゲズズゲ葦ザs httpsr｠｠doisorg｠ゲグsザザズジ｠mepsゲ葦ザゲズズ

Dongp Ys Wsp Mi旭旭erp Ls Psp Sandersp Js Gsp ｹ Somerop Gs Ns ｪゴググ芦ｫs Heat shock protein ゼグ ｪHspゼグｫ expression in four 旭impets of the genus

科架 科 | 科ゲゾゾザFunctional EcologyCLARK ET AL

Lottia Interspecific variation in constitutive and inducible synthesis

corre旭ates with in situ exposure to heat stresss Biological Bulletinp 215p ゲゼザゲ芦ゲs httpsr｠｠doisorg｠ゲグsゴザグゼ｠ゴズジゼグ葦ゾ芦

Farahp Ms Esp ｹ Ambergp Ds Cs ｪゴググゼｫs Conserved actin cysteine residues are oxidative stress sensors that can regu旭ate ce旭旭 death in yeasts Molecular Biology of the Cellp 18p ゲザズゾゲザ葦ズs httpsr｠｠doisorg｠ゲグsゲグゾゲ｠mbcE06-08-0718

Fie旭dsp Ps Asp Zuzowp Ms Jsp ｹ Tomanekp Ls ｪゴグゲゴｫs Proteomic responses of blue mussel (Mytilusｫ congeners to temperature acc旭imations Journal

of Experimental Biologyp 215p ゲゲグ葦ゲゲゲ葦s httpsr｠｠doisorg｠ゲグsゲゴジゴ｠jeb062273

Forsmanp As ｪゴグゲズｫs Rethinking phenotypic p旭asticity and its conse-

quences for individua旭sp popu旭ations and speciess Heredityp 115p ゴゼ葦ゴ芦ジs httpsr｠｠doisorg｠ゲグsゲグザ芦｠hdysゴグゲジsゾゴ

Freitesp Lsp Labartap Usp ｹ Fern史ndezReir趣zp Ms Js ｪゴググゴｫs Evo旭ution of fatty acid profiles of subtidal and rocky shore mussel seed (Mytilus gallo-

provincialisp Lmksｫs Inf旭uence of environmenta旭 parameterss Journal of

Experimental Marine Biology and Ecologyp 268p ゲ芦ズゴグジs httpsr｠｠doisorg｠ゲグsゲグゲ葦｠Sググゴゴグゾ芦ゲｪグゲｫググザゼゼX

Ga旭hardop Rs Ssp Hastingsp Ps Jsp ｹ Rosenbergp Ss Ms ｪゴググゼｫs Mutation as a stress response and the regu旭ation of evo旭vabi旭itys Critical Reviews

in Biochemistry and Molecular Biologyp 42p ザゾゾジザズs httpsr｠｠doisorg｠ゲグsゲグ芦グ｠ゲグジグゾゴザグゼグゲ葦ジ芦ズグゴ

Graceyp As Ysp ｹ Connorp Ks ｪゴグゲ葦ｫs Transcriptiona旭 and metabo旭omics characterisation of spontaneous metabolic cycles in Mytilus cali-

fornianus under subtidal conditions Marine Genomicsp 30p ザズジゲs httpsr｠｠doisorg｠ゲグsゲグゲ葦｠jsmargensゴグゲ葦sグゼsググジ

Grantp Ws Wsp Cherryp Ms Isp ｹ Lombardp As Ts ｪゴグゲグｫs A cryptic species of Mytilus ｪMo旭旭uscap Biva旭viaｫ on the west coast of South Africas South

African Journal of Marine Sciencep 2p ゲジゾゲ葦ゴsHa旭pinp Ps Msp Mengep Bs Asp ｹ Hofmannp Gs Es ｪゴググジｫs Experimenta旭

demonstration of plasticity in the heat shock response of the inter-

tidal mussel Mytilus californianus Marine Ecology Progress Seriesp 276p ゲザゼゲジズs httpsr｠｠doisorg｠ゲグsザザズジ｠mepsゴゼ葦ゲザゼ

Hardcast旭ep Ts Jsp ｹ Ke旭旭yp Ks As ｪゴグゲグｫs BaySeqr Empirica旭 Bayesian meth-

ods for identifying differentia旭 expression in sequence count datas BMC Bioinformaticsp 11p ジゴゴs httpsr｠｠doisorg｠ゲグsゲゲ芦葦｠ゲジゼゲゴゲグズ 11-422

Har旭eyp Cs Ds Gsp Dennyp Ms Wsp Machp Ks Jsp ｹ Mi旭旭erp Ls Ps ｪゴググゾｫs Therma旭 stress and morpho旭ogica旭 adaptations in 旭impetss Functional Ecologyp 23p ゴゾゴザグゲs httpsr｠｠doisorg｠ゲグsゲゲゲゲ｠jsゲザ葦ズゴジザズsゴググ芦sグゲジゾ葦sx

Harperp Es Msp C旭arkp Ms Ssp Hoffmanp Js Isp Phi旭ippp Es Es Rsp Peckp Ls Ssp ｹ Mor旭eyp Ss As ｪゴグゲゴｫs Iceberg scour and she旭旭 damage in the Antarctic biva旭ve Laternula elliptica PLoS ONEp 7p eジ葦ザジゲs httpsr｠｠doisorg｠ゲグsゲザゼゲ｠journa旭sponesググジ葦ザジゲ

Hinchp Ss Gsp Bai旭eyp Rs Csp ｹ Greenp Rs Hs ｪゲゾ芦葦ｫs Growth of Lampsilis ra-

diata ｪBiva旭viar Unionidaeｫ in sand and mudr A reciproca旭 transp旭ant experiments Canadian Journal of Fisheries and Aquatic Sciencep 43p ズジ芦ズズゴs httpsr｠｠doisorg｠ゲグsゲゲザゾ｠f芦葦グ葦ズ

Hoffmanp Js Isp Peckp Ls Ssp Hi旭旭yardp Gsp Zieritzp Asp ｹ C旭arkp Ms Ss ｪゴグゲグｫs No evidence for genetic differentiation between Antarctic 旭impet Nacella concinna morphotypes Marine Biologyp 157p ゼ葦ズゼゼ芦s httpsr｠｠doisorg｠ゲグsゲググゼ｠sググゴゴゼググゾゲザ葦グズ

Johannessonp Ksp ｹ Mikhai旭ovap Ns ｪゴググジｫs Habitat re旭ated ge-

netic substructuring in a marine snai旭 ｪLittorina fabalisｫ in-

vo旭ving a tight 旭ink between an a旭旭ozyme and a DNA 旭ocuss Biological Journal of the Linnean Societyp 81p ザグゲザグ葦s httpsr｠｠doisorg｠ゲグsゲゲゲゲ｠jsゲグゾズ芦ザゲゴsゴググザsググゴ芦芦sx

Johannessonp Ksp ｹ Tatarenkovp As ｪゲゾゾゼｫs A旭旭ozyme variation in a snai旭 (Littorina saxatilisｫ Deconfounding the effects of microhabitat and gene f旭ows Evolutionp 51p ジグゴジグゾs

Labartap Usp Fern史ndezReir趣zp Ms Jsp ｹ Babarrop Js Ms Fs ｪゲゾゾゼｫs Differences in physio旭ogica旭 energetics between intertida旭 and raft cu旭tivated mussels Mytilus galloprovincialis Marine Ecology Progress Seriesp 152p ゲ葦ゼゲゼザs httpsr｠｠doisorg｠ゲグsザザズジ｠mepsゲズゴゲ葦ゼ

Leadshamp Js Esp ｹ Gour旭ayp Cs Ws ｪゴググ芦ｫs Cytoske旭eta旭 induced apoptosis in yeast Biochimica amp Biophysica Actap 1783p ゲジグ葦ゲジゲゴs httpsr｠｠doisorg｠ゲグsゲグゲ葦｠jsbbamcrsゴググ芦sグゲsグゲゾ

Lip Hsp Ruanp Jsp ｹ Durbinp Rs ｪゴググ芦ｫs Mapping short DNA sequencing reads and ca旭旭ing variants using mapping qua旭ity scoress Genome Researchp 18p ゲ芦ズゲゲ芦ズ芦s httpsr｠｠doisorg｠ゲグsゲゲグゲ｠grsグゼ芦ゴゲゴsゲグ芦

Lockwoodp Bs Lsp Sandersp Js Gsp ｹ Somerop Gs Ns ｪゴグゲグｫs Transcriptomic responses to heat stress in invasive and native blue mussels

ｪgenus Mytilusｫr Mo旭ecu旭ar corre旭ates of invasive successs Journal

of Experimental Biologyp 213p ザズジ芦ザズズ芦s httpsr｠｠doisorg｠ゲグsゲゴジゴ｠jeb046094

Moranp Psp MarcoRiusp Fsp Megiasp Msp Cove旭oSotop Lsp ｹ PerezFigueroap As ｪゴグゲザｫs Environmenta旭 induced methy旭ation changes associated with seawater adaptation in brown trout Aquaculturep 392p ゼゼ芦ザs httpsr｠｠doisorg｠ゲグsゲグゲ葦｠jsaquacu旭turesゴグゲザsグゴsググ葦

Mor旭eyp Ss Asp C旭arkp Ms Ssp ｹ Peckp Ls Ss ｪゴグゲグｫs Depth gradients in she旭旭 morpho旭ogy corre旭ate with therma旭 旭imits for activity and ice distur-bance in Antarctic 旭impetss Journal of Experimental Marine Biology amp

Ecologyp 390p ゲズs httpsr｠｠doisorg｠ゲグsゲグゲ葦｠jsjembesゴグゲグsグジsグジグMor旭eyp Ss Asp Lemmonp Vsp Obermu旭旭erp Bs Esp Spicerp Js Isp C旭arkp Ms Ssp

ｹ Peckp Ls Ss ｪゴグゲゲｫs Duration tenacityr A method for assessing ac-

c旭imatory capacity of the Antarctic 旭impetp Nacella concinna Journal

of Experimental Marine Biology amp Ecologyp 399p ザゾジゴs httpsr｠｠doisorg｠ゲグsゲグゲ葦｠jsjembesゴグゲゲsグゲsグゲザ

No旭anp Cs Ps ｪゲゾゾゲｫs Sizep shape and she旭旭 morpho旭ogy in the Antarctic limpet Nacella concinna at Signy Is旭andp South Orkney Is旭andss Journal of Molluscan Studiesp 57p ゴゴズゴザ芦s httpsr｠｠doisorg｠ゲグsゲグゾザ｠mollus572225

Peckp Ls Ss ｪゴグゲ葦ｫs A co旭d 旭imit to adaptation in the seas Trends in

Ecology amp Evolutionp 31p ゲザゴ葦s httpsr｠｠doisorg｠ゲグsゲグゲ葦｠jstreesゴグゲズs 09014

Peckp Ls Ssp Heiserp Ssp ｹ C旭arkp Ms Ss ｪゴグゲ葦ｫs Very s旭ow embryonic and 旭arva旭 deve旭opment in the Antarctic 旭impet Nacella po-

laris Polar Biologyp 39p ゴゴゼザゴゴ芦グs httpsr｠｠doisorg｠ゲグsゲググゼ｠s00300-016-1894-1

Peckp Ls Ssp Mor旭eyp Ss Asp Richardp Jsp ｹ C旭arkp Ms Ss ｪゴグゲジｫs Acc旭imation and therma旭 to旭erance in Antarctic marine ectothermss Journal of

Experimental Biologyp 217p ゲ葦ゴゴs httpsr｠｠doisorg｠ゲグsゲゴジゴ｠jebsグ芦ゾゾジ葦Powe旭旭p As Ws Bs ｪゲゾズゲｫs Antarctic and sub Antarctic mo旭旭uscar Pe旭ecypoda

and Gastropodas Discovery Reportsp 26p ジゾゲゾ葦sReyna Lopezp Gs Esp Simpsonp Jsp ｹ RuizHerrerap Js ｪゲゾゾゼｫs Differences

in DNA methy旭ation patterns are detectab旭e during the dimorphic transition of fungi by amp旭ification of restriction po旭ymorphismss Molecular amp General Geneticsp 253p ゼグザゼゲグs

Serafinip Lsp Hannp Js Bsp Ku旭tzp Dsp ｹ Tomanekp Ls ｪゴグゲゲｫs The proteomic response of sea squirts ｪgenus Cionaｫ to acute heat stressr A g旭oba旭 perspective on the thermal stability of proteins Comparative amp

Biochemical Physiology Dp 6p ザゴゴザザジsShangp Xs Ysp Sup Js Gsp Wanp Qs Ysp ｹ Sup Js Js ｪゴグゲズｫs CpA｠CpG methy旭ation

of CiMDAズ possesses tight association with the resistance against GCRV and negative旭y regu旭ates mRNA expression in grass carpp Ctenopharyngodon idella Developmental amp Comparative Immunologyp 48p 芦葦ゾジs httpsr｠｠doisorg｠ゲグsゲグゲ葦｠jsdcisゴグゲジsグゾsググゼ

Somerop Gs Ns ｪゴグゲグｫs The physio旭ogy of c旭imate changer How potentia旭s for acc旭imatization and genetic adaptation wi旭旭 determine wwinnersx and losers Journal of Experimental Biologyp 213p ゾゲゴゾゴグs httpsr｠｠doisorg｠ゲグsゲゴジゴ｠jebsグザゼジゼザ

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How to cite this artic旭er C旭ark MSp Thorne MASp King Mp Hipperson Hp Hoffman JIp Peck LSs Life in the intertida旭r Ce旭旭u旭ar responsesp methy旭ation and epigeneticss Funct Ecol

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