Posted on Authorea 19 Feb 2020 | CC BY 4.0 | https://doi.org/10.22541/au.158212862.25725074 | This a preprint and has not been peer reviewed. Data may be preliminary. euaino ee eae oidcino eoi iiina ela ee epnil o post-translational for up- responsible genes caused as temperatures well elevated as metabolism, species, division heat-sensitive lipid meiotic the of and induction In carbohydrate to energy, proteins. related opposing as genes ribosomal caused such of to induction regulation categories related Temperature functional genes temperatures. in further different and in to expression adaptation of heat. species’ patterns to a of responses oppo- ( component an opposing key proteins followed and heat-tolerant shock shared the heat both while species, of revealed growth, found expression species analyses population notably, We heat-sensitive transcriptomic low Most the Comparative and temperatures. with survival strategy. increasing response, high site heat of across in strategy expression a differences gene adopting cross-temperature comparing and by cross-species variation significant mecha- heat-sensitive physiological and molecular we of heat-tolerant demography, the basis related temperature-related but closely compare To in affects rates, experiments chionus temperature unknown. growth and life-table Environmental largely occurrence population cross-temperature are determine and performed change. alterations can life-spans these climate that underlying to altering trait nisms physiological response by essential including performance an nature, zooplankton is in stress succession thermal to temporal response species’ A Abstract mechanisms molecular potential warming. reveals global as and of to division reorganizations species adaptation two sweeping meiotic for the these activated of demonstrates in be induction work adaptation might to This that temperature proteins. related accompany modifications. ribosomal genes that histone to of functions post-translational related biological up-regulation for genes expression in responsible caused and of genes key temperatures metabolism, patterns as a elevated lipid well likely opposing species, and are caused heat-sensitive carbohydrate genes induction energy, two hsp the Temperature as the that In such showing in temperatures. categories different rates, different functional growth strikingly to further is population adaptation in (hsps) in low species’ proteins differences and shock mirrored a survival both heat responses of of revealed high hsp component expression analyses species, of notably, transcriptomic both Most strategy Comparative In heat. a strategy. species. to adopting opposite responses species an opposing followed and heat-sensitive and heat-tolerant shared cross-species the the significant with while found basis We growth, response, genetic population temperatures. heat the increasing examined in we across compare differences expression populations, To and same gene cross-temperature heat-tolerant these comparing unknown. Within related by largely variation sympatry. closely physiological in are in of occur alterations that experiments these species life-table rotifer underlying Brachionus cross-temperature mechanisms heat-sensitive performed molecular we in the life-spans succession demography, altering but temporal by temperature-related and performance rates, zooplankton occurrence growth affects determine temperature can population Environmental that and trait change. physiological climate essential to an response is including stress nature, thermal to response species’ A Abstract 2020 5, May 1 Paraskevopoulou Sofia rotifers Brachionus heat-sensitive versus heat-tolerant responses in transcriptomic and history life Temperature-dependent nvriyo Potsdam of University hsp oie pce htocri ypty ihnteesm ouain,w xmndtegenetic the examined we populations, same these Within sympatry. in occur that species rotifer epne irrddffrne npplto rwhrts hwn that showing rates, growth population in differences mirrored responses 1 lc Dennis Alice , 1 uta Weithoff Guntram , hsps 1 ssrknl ieeti h w pce.I both In species. two the in different strikingly is ) 1 n ap Tiedemann Ralph and , hsp ee r ieya likely are genes 1 Bra- Posted on Authorea 19 Feb 2020 | CC BY 4.0 | https://doi.org/10.22541/au.158212862.25725074 | This a preprint and has not been peer reviewed. Data may be preliminary. osqec feooia ieetain(oeo uisy ia,21) qai aaueavariety a use taxa as Aquatic selection 2012). divergent Gilad, via contributing & evolved genes Ruvinsky, been revealing (Romero, have in differentiation might useful ecological expression particularly of gene consequence are in 2003). a data Differences al., transcriptome plasticity. et (Wray expression, phenotypic plasticity phenotypic distribution to on underlying temporal acts mechanism their essential selection an in As is role expression a gene of plays Regulation likely tolerance 2018). temperature Weithoff, & that Tiedemann confirming (Paraskevopoulou, thus former, 2017). abundance between al., the and tolerance et of occurrence Zhang heat 2016; temporal on al., studies the et affect Wen 2010; and to spring, Ma, shown & have were Niu, occurrences constraints (Li, their of and studies temperature succession, several specifically, temporal in exhibit More temperature complex this to of related species been The 2016). al., et Papakostas species: the different four is nandoi to rotifer resolved Ramos-Rodr´ıguez, monogonont Montero-Pau, been freshwater recently 2007; studied Serra, best cases, & The these Melone 2017). In Fontaneto, Giordani, 2017). & over (Fontaneto, al., competition Serra G´omez, time et reduce 2011; & to or Zhang Ciros-P´erez, Serra specialties Tri- 2017; space ecological Michaloudi, Ge, different in (Papakostas, & evolved Suatoni, rotifers resources Wen, have in 2017; might Xi, phenomenon adapted, species Fontaneto, Xiang, common differentially similar 2013; & a morphologically of Abatzopoulos, is Serra co-occurrence locality & specialization, 2017; single Kappas of a antafilidis, drive al., evidence in to As et species likely 2006). on related Mills more Caccone, closely dependent 2002; & is less Snell Lunt, specialization seems conditions Rice, ecological diversification & Vicario, ecological that and Carvalho suggesting specific distribution (G´omez, Serra, Serra, factors, ubiq- large, to speciation & historical be be adapted Montero-Pau, and can to Carmona, barriers species capabilities assumed (Gabald´on, Fontaneto, geographical dispersal formerly cryptic salinity their complexes or comprise As Species (Fontaneto, type, to 2017). capability habitat 2017). found dispersal temperature, al., been high regarding et their have diversity, zoo- Mills their cryptic, generalists Among 2009; and often uitous Barraclough, high, conditions, 2008). and & their environmental (Segers, compounds Herniou of levels specific Kaya, because organic trophic to interest transfer higher particular adaptation to they of frequent phytoplankton) are as species rotifers ecosystems, (e.g. monogonont understanding plankton, producers aquatic Therefore, primary of how from components 2017). and energy environment important al., their are et to Zhang adapted Zooplankton change. have climate 2011a; species to al., how respond comprehending et may tem- for they Xiang essential Xi, species-specific Wen, is 2016; evolved 2013; boundaries have Xiang, thermal Abatzopoulos, might & & that succession Xue, Kappas, species Triantafyllidis, seasonal Zhang, Northington, similar Michaloudi, temperature-dependent & (Papakostas, genetically and specializations Chaloner among occurrence, perature documented Lamberti, temporal Hershey, well others impact while been 2008; temperature, can has (Cullum, of the this range limits in Importantly, broad variation temperature a great 2010). narrow dis- tolerate is can spatio-temporal and There species abundance, specific Many 2006). Parmesan, species have taxa. pa- 2013; affect aquatic environmental al., among can of et tolerance range profound and (Paaijmans thermal a particular performance, colonization has a habitat and temperature of and survival ecosystems, tolerance tribution, aquatic organism’s its In an to precipitation. on related and impact is salinity temperature, occurrence as species’ such a rameters scale, global a On INTRODUCTION that mechanisms that molecular functions potential warming. biological global reveals Keywords: to and of adaptation species reorganizations for two activated sweeping these be the might in demonstrates adaptation work temperature This accompany modifications. histone .calyciflorus B. , rcinsdorcas, Brachionus .clcflrss.s. calyciflorus B. rcinscalyciflorus Brachionus rpi pce ndffrn aiasi hn,with China, in habitats different in species cryptic and nsme Xage l,21;Zage l,21) oprtv laboratory Comparative 2017). al., et Zhang 2017; al., et (Xiang summer in rcinselevatus Brachionus .calyciflorus B. , rcinsfernandoi Brachionus rcinscalyciflorus Brachionus ..and s.s. 2 sn nertv aooy(ihluie l,2018; al., et (Michaloudi taxonomy integrative using , rcinscalyciflorus Brachionus .fernandoi B. hra dpain N-e,heat-stress RNA-seq, adaptation, thermal , .fernandoi B. es tit (s.s.), stricto sensu aesonhge heat-tolerance higher shown have pce ope hthas that complex species curn nwne and winter in occurring rcinsfer- Brachionus Posted on Authorea 19 Feb 2020 | CC BY 4.0 | https://doi.org/10.22541/au.158212862.25725074 | This a preprint and has not been peer reviewed. Data may be preliminary. eoe.Eey2htemtra niiul eetaserdit e elwt rs odsuspension. food fresh were with neonates well hatched new newly a any into and transferred were h individuals 12 maternal every the recorded and were 24h well Every reproduction new removed. and a Survival thoroughly into recorded. inspected neonate 10 were and of one x well, composed introducing microtitre (2 by suspension a sp. algal sub- started in a of were placed 1ml bearing experiments culture, adding females Life-table the single neonates. from 2007): hatched isolated (2004, for Weithoff were experiment. from egg the procedure asexual of the itaneous, initiation followed the basically before experiments collapse The culture to led always mortality high (20 assays n ek(tlattot orgnrtos eoesatn h xeiett lo o clmto,adto and acclimation, for allow to (20 experiment temperatures the supplied four starting was at highest Food before conducted the generations) were effects. for four maternal weeks) to potential (2 two reduce least period by adaptation (at acclimatization week longest gradual the one of with cultures, period (32 among a temperature varied to period exposed acclimation were the cultures 2 temperature experiments, the table increasing life the starting Before flasks gene- glass ITS1 in the cultures of 20 batch at and portion as species, 1972) maintained a algae two Lorenzen, were amplifying of & cultures combination by for (Guillard Stock conditions confirmed medium 2018). laboratory previously WC al., under containing was et reared identity (Paraskevopoulou were Species marker and years. tic Germany 10 Northern than from heat-tolerant more originate clones the Both of periments. clones reproducing =43.18 conditions asexually experimental table Lab-reared, life and culture Rotifer change. 2.1 climate of heat- scenarios temporal different among METHODS species’ by variation AND determines affected MATERIALS physiology their be how might investigating understand this species. and to two how important the and tolerance is between distribution, data thermal difference species transcriptomic its heat-sensitive in and collect survival, and involved We response tolerant examine physiological mechanisms species. to of two the basis experiments the genetic Identifying life-table between the use examine differences We to rate conditions. (RNA-seq) growth temperature population high former and the fecundity, to in mild species related under closely expression two between tolerance thermal calyciflorus in variation marked the investigate To 2012). Snell, & Shearer, Burns, Smith, among eukaryotes which particularly vary higher induction in rotifers, to of proteins In prokaryotes conditions the 1993). from and Lindquist, described involved hsp40 genes is & specific and (Parsell the mechanism, However, taxa 1999). conserved Hofmann, and of & kinases) old Induction (Feder (e.g., 1998). evolutionary proteins Nardai, Proh´aszka, an other & Soti, of Schnaider, is (Csermely, activity cytoskeleton the the regulate stabilize they and newly Furthermore, of proteins. folding folded 2005). correct incorrectly Bukau, the removing promote & and (Mayer aggregates proteins peptide of synthesized accumulation reduce They e.g. conditions. (kDa): adverse ( weights proteins molecular shock response different heat shock proteins. of for heat (families) on encoding groups studies genes Transcription several on changes. focused temperature with often have cope to mechanism physiological of Cryptomonas aiisices etsoksria,sgetn htteemyb oriainaogha shock heat among coordination be may there that suggesting survival, shock heat increase families o )adteheat-sensitive the and C) Hsp70s ° 4 ,23 C, pce ope Prseoolue l,21) ecmae iehsoydmgah n gene and demography life-history compared we 2018), al., et (Paraskevopoulou complex species el/l,t vi odlmtto.Frec eprtr n pce,a es 4individuals 24 least at species, and temperature each For limitation. food avoid to cells/ml), ncmiainwt te rtis lyavtlrl nsrs oeac n uvvlunder survival and tolerance stress in role vital a play proteins, other with combination in , ° ° ) fe ecigteeprmna eprtr,w anandtertfrclue for cultures rotifer the maintained we temperature, experimental the reaching After C). ,26 C, hsp40 p Cluecleto ¨tign tanSG2-0,wspoie weekly. provided was SAG-26-80), G¨ottingen, strain collection (Culture sp. ° )wr sd etidsvrltmst acclimatize to times several tried We used. were C) ok yegsial oregulate to synergistically works ° vr asutlrahn h xeietltmeaue eas fthis, of Because temperature. experimental the reaching until days 2 every C ooahdu minutum Monoraphidium .fernandoi B. ° ,23 C, ooahdu minutum Monoraphidium Hsp90s ° coeA0 CT A10; (clone ,26 C, 3 dlibitum ad lyas ao oei testlrne anyby mainly tolerance, stress in role major a also play ° ,32 C, Cluecleto ¨tign tanSAG-243-1) G¨ottingen, strain collection (Culture hsp70 Brachionus ° ne 68lgtdr htpro.Afood A photoperiod. light:dark 16:8 a under C ° ) hl for while C), hsps max al.For daily. satvt a hw in shown (as activity ’s .clcflrss.s. calyciflorus B. s9,hp7,hp0 hsp40 hsp60, hspP70, hsp90, .Ha hc rtisaedvddinto divided are proteins shock Heat ). =38.49 5x10 x (5 pce,mmeso the of members species, .calyciflorus B. .fernandoi B. .fernandoi B. o 5 )wr eetdfrorex- our for selected were C) el/l and cells/ml) coeIB CT IGB; (clone he temperature three lot 32 to also .. experiments s.s., .manjavacas B. Cryptomonas n small and , hsp70 hsp ° ,but C, genes and max B. ; Posted on Authorea 19 Feb 2020 — CC BY 4.0 — https://doi.org/10.22541/au.158212862.25725074 — This a preprint and has not been peer reviewed. Data may be preliminary. sn ETe oy()Bas n tadseiclbaiswr ul sn ETe ai Illumina Rapid NEXTflex using built were libraries strand-specific and Beads, 3 (A) from Poly enriched was NEXTflex USA). Scientific, Technologies, mRNA using (Agilent (ThermoFischer preparation, 2100 library spectrometer Bioanalyzer 20 Agilent transcriptomic 1000 using with For examined NanoDrop was performed a RNA total was using of step Quality estimated Germany). was elution concentration double RNA A Total instructions. manufacturer’s the RNeasy an 4 into at minutes 500 15 of for total A independent temperature. in room at reared individuals, TRIzol 1000 in Samples of of approximately exception comprised 200ml. the replicate, of with into Each flasks supernatant) species used. the and were 30 in temperature replicates a 2000 (remaining each at through rotifers from it the (200ml) centrifuged replicates transferring replicate and TRIzol before each medium, of debris, WC filtered 1ml other in we and filter collection (26 phytoplankton the treatment specimen pellet on For heat remained what high 1). re-suspended and (Figure exposure), decline) heat to temperatures adjusted high (20 Experimental water-baths and fernandoi control (23 control represent into B. treatment hours. to between flasks heat 4 order temperature mild the in for intermediate acclimated), placed results rotifers were We life-table replicates the clones the four both heat-exposed to create individuals. to according and 1000 flasks in selected temperature new were used than into experimental that more stock the species at initial containing algae to the medium each, two sub-sampled WC of 200ml we containing combination temperature, of flasks and food glass species same 1L each The For in provided cultures photoperiod. was batch light:dark experiments life-table 16:8 as cultivated under first degC were 20 RNA-seq isolation for RNA Samples and using collection, performed cultivation, were Sample analyses 2.3 statistical All Team). Wacker). Core increase normal Dr. (R of communication data, 3.4.1 rate (personal the intrinsic R script the of R an compare as distribution To using comparisons, normal 2007) for violated. (ANOVA; were used Variance homoscedasticity) were non- of and tests ( Analysis the residuals, parametric perform used the Non to we of p-values. test distribution assumptions treatments the Wilcox the for pairwise temperature correction of the and Bonferroni comparisons all species a pairwise linear Saveliev, For the with Walker, generalized used variance. among Leno, used was of (Zuur, fecundity we criteria analysis compare AIC one-way interaction, Kruskal–Wallis To the their on parametric based and 2009). the model temperature, Smith, estimate best-fitting further the & species, To temperatures. selected by We test. across fecundity (GLMs). log-rank and and models a species using lifespan pairwise across on analyzed survival effect were compare functions to survival rate between calculated the Differences intrinsic were The as curves 1974). survival lx offspring (Poole, Kaplan-Meier days, interval of age number in any the interval of as age end mx r=[?]e the the and until interval as ( start age increase defined the the population from was of were of alive beginning (x (mx) female the fecundity assay per at age-specific produced temperature individuals and surviving and (lx) of survival species proportion age-specific each (survival), span for life died. estimated average cohort of each variables of The individuals analysis all statistical until continued and and Computations dark 2.2 the in conducted was experiment The r ,te9%cndneitra a siae i ottapn ih19ieain Wihff&Wacker, & (Weithoff iterations 199 with bootstrapping via estimated was interval confidence 95% the ), -rx l x= mx *lx n 32 and ® ® Sadsoigte t-80 at them storing and LS ® ° iiKtclm Qae,Gray n rcee oRApeiiainacrigto according precipitation RNA to proceeded and Germany) (Qiagen, column Kit Mini 1 ofcltt hs eaain ete rnfre h ools,uprauosphase aqueous upper colorless, the transferred then We separation. phase facilitate to C Swr ooeie sn iseLzr(mn 0Z n eeicbtdovernight incubated were and 50HZ) (4min, Lyzer Tissue a using homogenized were LS o for C r a loetmtdfo hs aauigLtaseuto Lta 1907): (Lotka, equation Lotka’s using data these from estimated also was ) .calyciflorus B. μ dlibitum ad fclrfr a de oec ape n ape eecentrifuged were samples and sample, each to added was chloroform of l .. eprtr nwihtepplto rwhrt starts rate growth population the which in temperature s.s.; o ni N xrcin o N xrcin eue four used we extraction, RNA For extraction. RNA until C vr w asfrtowesbfr h N-e experiment. RNA-seq the before weeks two for days two every 4 o for C .fernandoi; B. μ fttlRAwt oy()capture (A) poly with RNA total of g .fernandoi B. 26 μ o o for C Ns-rewtreach. water RNase-free l ;tmeauei which in temperature C; tml et hr two where heat, mild at × o 0mntsto minutes 10 for g .calyciflorus B. μ sieve, m o for C s.s.; Posted on Authorea 19 Feb 2020 — CC BY 4.0 — https://doi.org/10.22541/au.158212862.25725074 — This a preprint and has not been peer reviewed. Data may be preliminary. ocpuesmlro otatn atrso xrsinbtentetoseis rhlgu ee were genes orthologous species, two the between Weijun, expression (Luo, of “pathview” patterns Team). 2012), Core contrasting He, (R or packages & similar R Han, using 2016) capture Wang, performed (Wickham, To was (Yu, “ggplot2” visualization “clusterProfiler” and and 2009), 2013), Analyses Cory, differen- al., 0.05. & considered below et Brouwer, were (FDR) Pathways (Luo clustered pathways. rate were “gage” discovery biochemical performed. pathway false the was KEGG in a analysis same association at Okuda, expression the expressed their pathway Itoh, to tially differential (Moriya, on belonging a genes analysis based and pathway expression, genes pathway together KEGG KEGG clusters Encyclope- for whole which Kyoto server estimate online 2007), To automatic the (KEGG) Kanehisa, using Genomes & level Yoshizawa, and gene-pathway the Genes separately. at of species expression dia each differential for categorized counts All further log-transformed testing. We of multiple (PCA) for analysis correct component to ( principal 0.05 non-redundant two-dimensional of NCBI threshold 1e the cutoff against (FDR) (e-value annotated rate blastx were discovery (DEGs) false genes a expressed used differentially We 1). (Figure heat) vs. For 1). (Figure eentpeeti tlat2rpiae.T xmn nr-pce eprtr pcfi xrsinpattern expression specific temperature intra-species examine To ( replicates. genes vs. separately. 2 count species low least two count removed the at we estimated in analyzed analysis in the and expression present account passed differential 2014) not the we into Anders, were for & expression, taken model Huber, gene length the (Love, differential build gene DESeq2 To detect to effective To package tximport with tximport from 2016). files The matrices Robinson, package. quantification & tximport gene-level Love, the estimates from (Soneson, with quantification matrices Gene-level R/Bioconductor count into 2011). Dwey, imported produces & were (Li RSEM program by RSEM produced the incorporating “align software the 2011) ran al., we pathways expression and quantify genes To expressed differentially of Identification to 2.5 search blastn a the of since performing sequences bias, by a 28S reads and RNA the 18S ribosomal utilize remove of not to consisting used database further local was a script custom same blastn The the the ( to using database was which, algae hit script local a perl to custom either s.s. contigs a all used assigns minutum 2015), we raphidium al., contamination, et possible Camacho (ncbi-blast-2.6.0; filter algorithm To and species. (before for respective minimum quality created and Read were 20 2011). of transcriptomes assembled Usadel, threshold were 2010). Read & quality reads (Andrews, Lohse, Short mean v0.11.5 Processed (Bolger, FastQC a NCBI v0.36 using with the Trimmomatic assessed window low was using in sliding quality-filtering) and bp bp deposited after trimmed 36 5 were been of a sequences have length using Adapter read data filtered SRR10426055-76). system, were Raw sequencing (SRA: reads HiSeq4000 numbers quality China). Illumina accessions an the Kong, using under (Hong reads Archive (PE) Novogene paired-end by bp, 150 performed as sequenced were Libraries annotation was and control assembly quality sequencing, and USA). Transcriptome Germany) Technologies, 2.4 (Invitrogen, (Agilent instructions. Kit 2100 manufacturer’s Assay Bioanalyzer HS to Agilent dsDNA using according Qubit performed USA) using quantified 16 Scientific, were in Libraries (Bioo performed was Kit elution Prep Final Library RNA-Seq Directional 26 .calyciflorus B. 23 eoeasml Kme l,21) rncit eeol sinda frtfroii hntetop the when origin rotifer of as assigned only were Transcripts 2018). al., et (Kim assembly genome o (control C o (control C .clcflrss.s. calyciflorus B. .calyciflorus B. .fernandoi B. .fernandoi B. vs. .. epromdpiws otat ihntemdli h olwn obntos 20 combinations: following the in model the within contrasts pairwise performed we s.s., vs. , hayooa reinhardtii Chlamydomonas idha) 20 heat), mild idha) 20 heat), mild -10 .W sesdoealtmeauedpnetpten fepeso ypotn a plotting by expression of patterns temperature-dependent overall assessed We ). iial epromdpiws oprsn ntefloigcmiain:20 combinations: following the in comparisons pairwise performed we similarly ed ol o a swl oterfrnea hs from those as reference the to well as map not would reads ..gnm n h i-cr anoe ace otenx pce was species next the to matches over gain bit-score the and genome s.s. enovo de .calyciflorus B. eeec eoefrteaaye rsne ee sti ol introduce would this as here, presented analyses the for genome reference μ feuinbffradaPRapicto f1 ylswsperformed. was cycles 14 of amplification PCR a and buffer elution of l vs. vs. 32 and o ihTiiyv251(rbere l,21) eaaereference Separate 2011). al., et (Grabherr v.2.5.1 Trinity with 26 (control C estimate o (control C ..and s.s. and, 5 vs. Cryptomonas bnac.l citi rnt ... Gahr et (Grabherr v.2.5.1 Trinity in script abundance.pl” Brachionus vs. .fernandoi B. ihha) n 26 and heat), high ihha) n 23 and heat), high pce onoddfo CI edid We NCBI. from downloaded species p rt h respective the to or sp) sn l ed eeae o the for generated reads all using , vs. 32 vs. o (mild C 26 < nr .calyciflorus B. 0 n ee that genes and 10) o (mild C aaaeusing database ) .calyciflorus B. vs. ihheat) high vs. Mono- > high 100. s.s.. Posted on Authorea 19 Feb 2020 — CC BY 4.0 — https://doi.org/10.22541/au.158212862.25725074 — This a preprint and has not been peer reviewed. Data may be preliminary. dnie sn rhfidr(ms&Kly 05.Fo hs ecmae xrsino rhlgu genes orthologous of expression compared we this, From 2015). Kelly, between & (Emms Orthofinder using identified h iehsoytat uvvl eudt n h eutn ouaingot aeo ohseiswere comparisons species mild survival Cross-species both to control S1). of Table from in 20 rate available reduced p-values at growth significantly all differences 1A, was population significant (Figure survival heat revealed resulting species, high to the both mild For and from and fecundity temperature. by survival, affected traits strongly history life heat The to responses history a further Life using for 3.1 pathways, meaningful KEGG biologically any (FDR=0.05). were RESULTS in positives enrichment false that 3. for functional patterns correcting contrasting for chose and and checked Test we shared were Exact Fisher both this, groups/clusters in These Within (well-correlated) analysis. co-expressed species. consistently between are patterns that identified are (groups) .. 20 s.s.: calyciflorus than vs. ihgnsecdn o N oyeae n -ctlrneae en prgltda ihha.Genes heat. high at up-regulated being N-acetyltranferases and polymerases induction RNA temperature for different showed encoding genes In above genes transition. The (26 this with transferases. and mild in proteins between temperature histone of expressed polymerases, RNA effect differentially strong genes (Figure a 64 is pattern the there opposite that the data mild found rate comparison than growth we pairwise (1268) heat-sensitive on heat the focused mild in We and contrast, control In between (64). number expressed 3B). heat greater differentially 3A, high a genes and captured of we mild number between species greater both a In captured control heat-sensitive. we comparison the pairwise in than DEGs species of two- heat-tolerant the a in by (DEGs) species 20 each from ° replicates in which temperature-dependent in heat-tolerant, showed responses analysis species, the This sensitive temperature-dependent for PCA. a replicates for of of components principal data separation two first expression the of the plot dimensional examined first We expression gene differential (Table Cross-temperature libraries 3.3 among 1. reads Table in of found numbers the be equal about approximately Information with S3). respectively, reads, PE quality-filtered the of Sequencing transcriptomics Comparative 3.2 26 at maximum 23 its until temperature heat-sensitive However, the temperatures. for tested ( all increase at population survival, rate of (GLM: rate temperature intrinsic The on dependent significantly p were survival, (GLM: fecundity and survival p both that revealed heat-sensitive 26 at observed were differences omdasprt lse Fgr 2.Oeal efudagetrnme fdffrnilyepesdgenes expressed differentially of number greater a found we Overall, S2). (Figure cluster separate a formed C < o .0;fcniy p fecundity, 0.001; ,addcesda 26 at decreased and C, 26 .calyciflorus B. o o ,p C, C .fernandoi B. vs. ..and s.s. < .fernandoi B. .0;23 0.001; 32 .calyciflorus B. < o ,p C, .fernandoi B. .0;fcniy =.0)(iueSB S1C). S1B, (Figure p=0.001) fecundity, 0.001; . Fgr A al 1.Fcniyaayi ihteKWts eeldta both that revealed test K-W the with analysis Fecundity S1). Table 1A, (Figure s.s < .fernandoi B. and .0) pce GM uvvl p survival, (GLM: species 0.001), < o .0;23 0.001; C .calyciflorous B. o (Bcal26 vs. C. enovo de o ,i hc h heat-tolerant the which in C, 26 a infiatlwrfcniya h ihs moe et( heat imposed highest the at fecundity lower significant a had ..and s.s. o ,p004 iueSA al 1.Ars h w pce,significant species, two the Across S1). Table S1A, Figure p=0.004, C, o vs. C Fgr 2B) (Figure o sebis sebe ngns n EGK emasgmn can assignment term KO KEGG and unigenes, assembled assemblies, (p C o vs. .I contrast, In C. fr6 p Bfer26, vs. .fernandoi B. vs. r r < ihha Fgr A B.Hwvr nteha-oeatspecies, heat-tolerant the in However, 3B). 3A, (Figure heat high 32 using a lashge o h heat-tolerant the for higher always was a bv eofrbt pce,idctn oiiegrowth positive a indicating species, both for zero above was ) o .0)ad23 and 0.001) .calyciflorus B. ihha nbt pce sw a vdnefo population from evidence had we as species both in heat high o n 23 and C ,p001 26 p=0.001; C, Clust . < For .0,Fgr 1,TbeS) eeaie iermodels linear Generalized S2). Table S1A, Figure 0.001, 6 rncitmsgnrtd602236ad358,527,278 and 670,242,336 generated transcriptomes .calycifloryus B. AuJmu el,2018) Kelly, & (Abu-Jamous o o r lsee oehr hl l ape rae t26 at treated samples all while together, clustered C )adhg (32 high and C) < eandcntn in constant remained o .0;fcniy =.0) n hi interaction their and p=0.006), fecundity, 0.001; p00) with (p=0.08), C .calyciflorus B. ..Ti eaainwsls la o h heat- the for clear less was separation This s.s. o C vs. 32 s.s, o o ,p=0.002; C, ..hdhge eudt hnthe than fecundity higher had s.s. )ha,wr ee noigfor encoding genes were heat, C) r nrae ihteices of increase the with increased .fernandoi B. .fernandoi B. .calyciflorus B. .calyciflorus B. . In .fernandoi B. Clust, uvvn longer surviving rm20 from .calyciflorus B. eeclusters gene .. among s.s., ..than s.s. 20 : o to C o B. C Posted on Authorea 19 Feb 2020 — CC BY 4.0 — https://doi.org/10.22541/au.158212862.25725074 — This a preprint and has not been peer reviewed. Data may be preliminary. epne(S)i ohseis nbt pce,w on infiatdw-euaino etshock heat 4B). of 4A, Figure down-regulation 1B, significant for (Figure encoding a maximized Genes found was we for rate heat-tolerant, growth encoding the species, population genes both where general, In temperatures In species. at genes both protein in in (HSR) expression Response response of Shock patterns Heat examined in S4B). We (Figure genes temperatures expressed and higher differentially ubiquitin-ligase at Cross-temperature E3 up-regulated for 3.4 were encode polymerase genes RNA These were comparisons. proteins of genes heat-sensitive pairwise mediator ribosomal all related the a in In for proteases expressed S4A). while differentially encoding were (Figure temperatures, genes that temperatures lower these, higher at down- at Among up-regulated tolerant or up-regulated up- were comparisons. heat These either S-transferase comparisons. pairwise presenting the glutathione pairwise temperature all In and to three in responsive increase. the most expressed temperature all the differentially in are with they expressed along as differentially importance regulation are high that of H4K20. are genes H3K79, genes on H3K4, S3B). focused In as (Figure S3A). further such heat We (Figure proteins with heat methyltranferase up-regulated high histone were several at genes for regulated above down- encoding All or genes found up- we either heat, were family H2B as heat-sensitive such histones for encoding eedffrnilyepesdol in only expressed differentially were prgltda idha,addw-euae gi ne ihha.I otat for contrast, species. In two heat. the high in under again expression down-regulated of and patterns heat, heat-tolerant opposing mild the at with from up-regulated genes in genes contained enriched C6, C7) cluster significantly and In were (C6 pathways clusters were KEGG Two and Five 01100). species two (p Gene 6). the relevant. group between (Figure biologically C1 pattern appear temperature the expression patterns same increasing their the with because had these that up-regulated orthogroups of 150 three contained just C1 on with cluster focused Analysis have co-expression We for groups. searched S9). we gene (Figure species, two orthologous the between in expression of patterns “ribosome” patterns contrasting or in similar involved capture To genes groups gene while S8). co-expression Figure heat, 5B, Cross-species high (Figure 3.6 under species Genes up-regulated this heat. high for also under down-regulated were down-regulation were were pathway pathway metabolism meiosis lipid and in carbohydrate involved signal metabolism , to to to related related mainly pathways pathways while related of heat-sensitive, pathways down-regulation the of and heat-tolerant for up-regulation metabolism the caused lipid for heat and transduction high metabolism general, carbohydrate In as such S6). S5, for Figure exposure 5A, heat mild under down-regulated fernandoi (32 calyciflorus B. for encoding vrl,ml etrsle ndw-euaino ahasrltdt eei nomto rcsigin processing information genetic to related KEGG pathways whole of examined down-regulation control we in comparisons, pathways, resulted heat-tolerant, pairwise gene- heat two of mild in level expression Overall, pathway the KEGG at whole vs. patterns compared We contrasting expression. and pathway shared capture expression To pathway KEGG differential Cross-temperature 3.5 idha n mild and heat mild o ) olwn h nuto atr of pattern induction the following C), oeseicly ahas“iooe,“rtaoe,ad“xdtv hshrlto”were phosphorylation” “oxidative and “proteasome”, “ribosome”, pathways specifically, More . hsp90 .fernandoi B. .calyciflorus B. .. ee noigfor encoding genes s.s., < .5 D=.5,icuiggnsblnigt oemtblcptwy KG pathway: (KEGG pathways metabolic core to belonging genes including FDR=0.05), 0.05, .calyciflorus B. eawr prgltdudrtehgetipsdtmeauergm nbt pce.In species. both in regime temperature imposed highest the under up-regulated were beta hsp vs. fernandoi B 7and 27 ihha,bcueteecmaiosrpeetasews eprtr induction. temperature stepwise a represent comparisons these because heat, high mn h 1 ieetal xrse ewe id(23 mild between expressed differentially 310 the among , .. n prglto fmtblcrltdptwy nteheat-sensitive, the in pathways related metabolic of up-regulation and s.s., hsps hsp ..ada h ihs eprtr o h heat-sensitive, the for temperature highest the at and s.s. .calyciflorus B. a ihrepeso ttelws eprtr ramn (20 treatment temperature lowest the at expression higher had 0floe hspten ee noigfor encoding Genes pattern. this followed 70 nwihptwy eae osga rndcinwr up-regulated, were transduction signal to related pathways which in , .calyciflorus B. hsp20 hsp B.calyciflorus hsp90 ee oeaut hi pcfi otiuint h etshock heat the to contribution specific their evaluate to genes eeas prgltdudrtehgetipsdtemperature imposed highest the under up-regulated also were .calyciflorus B. ea(iue4A). (Figure beta ..(iue5,Fgr 7.Teopst a observed was opposite The S7). Figure 5B, (Figure s.s. ..adfloe h nuto atr of pattern induction the followed and s.s. Clust 7 ..hdlwepeso tcnrlcniin,were conditions, control at expression low had s.s. eeldtepeec f8gn groups/clusters gene 8 of presence the revealed ..adu-euae for up-regulated and s.s. calyciflorus B .fernandoi, B. hsp .. hr ee2 genes 23 were there s.s., .fernandoi B. 10, .fernandoi B. hr eetogenes two were there o )adhg (26 high and C) hsp 0 and 40, hsp .fernandoi. B. 0 Genes 70. ee in genes , (Figure o hsp )for C) o C) B. 60 Posted on Authorea 19 Feb 2020 — CC BY 4.0 — https://doi.org/10.22541/au.158212862.25725074 — This a preprint and has not been peer reviewed. Data may be preliminary. ieecsi iehsoisbtensbigseiso the into of translate species also sibling might between their histories which further both life tolerance, results temperatures, in Our temperature of Differences different. range in is a specialized genes survive are heat-stress can specializations. species representative habitat species these of 2017). both that expression al., although and corroborates et that rate Zhang shown growth have 2016; We population al., temperature 2017). et to al., Wen adaptation et 2010; al., an densities et reflect their however, of (Li might temperatures, of temperature of which densities range ambient broad 2010) High to a al., survive relative can considerably et and vary habitats Li natural their 2017; and in Both variation tolerance year al., fluctuations habitats. temperature rate the aquatic et broad in growth of rotifers, (Zhang occurring that periods In fluctuations found different 2017). suggested Fontaneto, in was been & dominance (Serra it has their & possible this, facilitate Serra, co-occurrence Gabald´on, on salinities which Montero-Pau, sympatric 2007; of Based makes species, range al. 2014). among wide et specializations Serra a (Lowe implies tolerated and differentially Walczynska species respond while 2013; rates - Carmona, growth that population found and their Previous species - of 2013). sibling Weithoff, complex between Kemp, & Lowe, constrains rotifer 2005; Laufenstein, salinity Weisse, (Stelzer, the environment 2011; in particular Enesco, a work & in Kauler grow to 2007; rotifers Montagnes, D´ıaz-Avalos, of & ability stressful. the too representing ( response, are rate conditions which growth above ( Population limit exposure a heat did to high hand up at other species, certain only the both a reduced on to significantly Fecundity, 2010). was up 2017). Wen, 32 Fecundity accelerated (Stelzer, & pattern. s.s., are resources temperature, same Ma, development ambient the Xi, food egg Zhang, follow increasing unlimited Ma, and Xi, not given juvenile with 2010; Xiang, result, limit, increases al., 2016; a temperature et temperature Xi, As critical Li body rates. & 2011; rotifer metabolic Chen, Enesco, nature, accelerates Tao, taxa & which ectothermic Jiang, rotifer (Kauler their Xiang, other both survival to in in 2010; on survival; Due studies Xiang, temperature on that corroborate & temperature of environment results Wen, of effect Our their Zhang, effect span. profound in profound life a a changes reduced showed found frequent to that we led to species temperature both exposed increasing In are species history. life habitats their variable impact temporally heat-sensitive may and in heat-tolerant living between Rotifers response & heat (DeBiasse history data response Life phenotypic transcriptomic with 4.1 the transcriptome combining testing by of stressor power species- environmental 2016). the and an Kelly, with demonstrates to shared associated This organism pathways both functional an species. key revealed of identified these has and in heat, This to adaptation experiments. response temperate in history expression gene life in resolved from patterns specific recently data the phenotypic from with between 2018) consistent responses al., temperature-dependent in et variation (Paraskevopoulou florus test species to heat-sensitive data and expression heat-tolerant and history life combined We repair. cluster, and this replication In (p and heat. systems, enriched high endocrine DISCUSSION significantly under and were nervous up-regulated pathways transduction, and signal 12 conditions heat-sensitive in heat of involved the mild number for and However, total C6 control heat. in a for high enriched low at significantly was down-regulated was expression and 04212) conditions pathway: heat (KEGG again mild pathway up-regulated and regulating heat longevity mild (p cluster The at down-regulated heat. were conditions, high control at under expression higher had C6 .fernandoi B. pce ope Ppksa ta. 06.W bevdatasrpoi epneta a largely was that response transcriptomic a observed We 2016). al., et (Papakostas complex species o C; < .5 D=.5.I lse 7 for C7, cluster In FDR=0.05). 0.05, .fernandoi, B. aebe eotddrn pigadwne vndw o4 to down even winter and spring during reported been have .calyciflorus B. 26 r scniee rx oeaut niomna pcaiain n stress and specializations environmental evaluate to proxy a considered is ) o ) ugsigta eudt smitie cosvral eprtrsin temperatures variable across maintained is fecundity that suggesting C), rcinsplicatilis Brachionus ..hv enrpre uigsme pt 32 to up summer during reported been have s.s. .calyciflorus B. .calyciflorus B. 8 sdpplto rwha rx oeaut the evaluate to proxy a as growth population used .calyciflorus B. ..gn xrsinwshg ne oto and control under high was expression gene s.s. ..and s.s. < .5 D=.5,aogwihpathways which among FDR=0.05), 0.05, .fernandoi B. pce ope aebe found been have complex species o Yn ta. 07 Zhang 2017; al., et (Yang C Brachionus o xeinetemperature experience ,wiehg densities high while C, .fernandoi B. .calyciflorus B. species .calyci- B. gene , Posted on Authorea 19 Feb 2020 — CC BY 4.0 — https://doi.org/10.22541/au.158212862.25725074 — This a preprint and has not been peer reviewed. Data may be preliminary. ih osiuecl tesfrwr dpe pce n tesrsos ih eiiitdudrthese under initiated be might response stress a and species adapted warm In for conditions. stress cold for constitute encoding might genes in in found while was temperature, species two our among heat-tolerant pattern expression reversed A Lee, among & common indicating Park, of heat-stress, Lee, expression high in under Seo, ones 2011; transformed non al., hsp20 the et to compared (Rhee survival temperatures that their in elevated increase to times 100 response of ( a bacteria induction Transformed et as that 2006). (Schoville suggests copepods of oysters This and Up-regulation copepods, 2016). In heat. species organisms. al., e.g. high aquatic et organisms, by in Lim mechanism aquatic induced common 2017; other a al., for in be et of might encoding also expression Kim genes dependent The their 2012; as species. temperature genes, al., both response, hsp be in heat other to regime to in temperature found contrast involvement higher In the specific temperatures. towards a up-regulated high supported at integrity patterns structural expression maintain and stress cellular the of (20 Proteins species. exposure heat-sensitive heat-tolerant temperature the express the of in for to end growth: stress lower found optimal the cold related at been of closely induced study, have temperature were the present heat genes outside the tolerate hsp induced In fernandoi, to were 2013). B. ability genes al. hsp their et specifically, in (Bedulina More differences habitat with warmer up-regulated a species heat, amphipods from to the exposed species as when to such habitat organisms cold aquatic a from other in cold also to growth.differently adapted differentially optimal of species for related expression zone’ Closely ‘comfort study, the present outside the are temperature) In (here, 2008). when that al., low showing et was species, Jong rate growth (De Population stress 1999). osmotic Hofmann, and & hsp (Feder pathogens, eukaryotes metals, and ( heavy prokaryotes protein both shock of in heat Induction present of induction mechanism the conserved involves evolutionary which heat-sensitive Response, and Shock heat-tolerant Heat in between supporting conditions, response abiotic, environmental histories different shock and life for Heat biotic that specialized 4.2 stressors, and indicate temperature. multiple diverged findings to ecologically to above regard are adapted with All species differentially particular survival. the are high that species and idea growth the sibling two population these low with temperature, of of our across increased to strategy to consistent According a respond are species. to with strategies the production differences different between egg observed evolved differentiation have and represent that calyciflorus species times, two really demonstrated developmental our they results, they juvenile i.e., Lemmen, fitness (24 corollary, and species, Zhang, conditions within egg a of temperature clones as study As tested and such recent species. traits food A these history stable 2014). between life under al., in performed et (Wang differences (2019) showed risk Declerck predation and or/and Papakostas, Zhou, competition to response in eae ee irrdmaue hne ntepplto rwhrt cosatmeauegradient. temperature a across rate growth population the in changes measured mirrored genes related Br-hsp20 ee was genes dpigalf taeyo ihpplto rwhadlwsria,a poe to opposed as survival, low and growth population high of strategy life a adopting hsps .calyciflorus B. pcfial otiue oicesdtemltlrne(hee l,21) prglto of Up-regulation 2011). al., et (Rhee tolerance thermal increased to contributes specifically .calyciflorus B. Brachionus hsp90 hsp20 on to found a encnetdt eea tescniin uha xouet xrm temperatures, extreme to exposure as such conditions stress several to connected been has ee eeidcdb et hl nheat-tolerant in while heat, by induced were genes hsps .fernandoi B. aiysret nraeteaalbecaeosi h el nodrt eoe from recover to order in cells the in chaperons available the increase to serve family eet ellrdfnemcaimi epnet ieetsrsosta ih be might that stressors different to response in mechanism defense cellular a reflects loices eitnet xdtv tes(hee l 01.Psil,a increase an Possibly, 2011). al. et (Rhee stress oxidative to resistance increase also ee r nedpr faseis tesrsos,we niomna conditions environmental when response, stress species’ a of part indeed are genes species. .. ee noigfor encoding genes s.s., .. h aeidcinptenwith pattern induction same the s.s., shrci coli Escherichia oad h ihrtmeaue tsesta eprtrssc s20 as such temperatures that seems It temperature. higher the towards hsp hsp20 ee eeu-euae.Ti atr a osseti both in consistent was pattern This up-regulated. were genes xrsigthe expressing ) ee a enrpre rvosyfo other from previously reported been has genes hsp70 vs. 9 amhbtt aebe on oexpress to found been have habitats warm hsp .calyciflorus B. eesgicnl prgltdtwrstelower the towards up-regulated significantly were ee led tlwrtmeaue compared temperatures lower at already genes rcinshsp20 Brachionus o ) niaigta 20 that indicating C), hsp70 uingmau.S Eulimnogammarus. .calyciflorus B. .. ee encoding genes s.s., ee olwdgnsecdn for encoding genes followed genes hsp Brachionus hsp90 ee,i elkonand known well a is genes, ) ( Br-hsps20 ..temjrt of majority the s.s. eeaogwt heat with along gene hsp70 hsp90 hsp o species a led be already may C ee differently. genes eisoriginated pecies hsp20 and eehsbeen has gene eehda had gene ) .fernandoi B. hsp90 Brachionus Brachionus hsp27 hsp eealso were genes were o In . hsp C), o B. C Posted on Authorea 19 Feb 2020 — CC BY 4.0 — https://doi.org/10.22541/au.158212862.25725074 — This a preprint and has not been peer reviewed. Data may be preliminary. l,21;Pdasy&Smr,20;Shvlee l,21;Tubn ta. 00.Rbsmlpoenrelated protein Ribosomal et 2010). al., (Lim et increase Truebano temperature tolerant 2012; upon al., heat parti- suppressed et Schoville or in to 2004; induced times genes, Somero, either several & been RNA mentioned Podrabsky have ribosomal been 2016; they al., have of and proteins response coordination stress Ribosomal requiring in production. process cipate protein energy-demanding ribosomal and and complex (rRNA) a is heat-sensitive biogenesis and availableRibosome heat-tolerant allocate between to response order in Ribosomal pathways) 4.4 ATP related of metabolic production of survival. rapid majority to for the resources need of species down-regulation a heat-sensitive by suggests (indicated the This while 2017). heat, Zhou, high & Apparently, Wang, fish, temperatures. teleost Liu, increasing a under Gao, in for (Yang, found required also exposure was are heat metabolism that carbohydrate to metabolites related mild mirabilis of genes from of up-regulated range heat were heat-tolerant, wide with metabolism regulation in or- carbohydrate a heat and acids an produce lipid fatty high of to to into to aspects related digestion essentials Genes all during survival. the down nearly and broken providing development affect are sugars, that carbohydrates protective simple and processes a lipids and conserved as 2016). consumed highly acts al, The are likely biology. et ganism’s (Lim metabolism stress tolerance oxidation carbohydrate to heat of and according enhances repression expression Lipid potentially their that and in indicates cells diversity This the with exposure. of down-regulated, mechanism 35 heat were genes of to above duration up the the elevation of both in temperature oyster, differed to Pacific also genus response genes the GST in of of down-regulated regulation species 14 Transcriptional on 2019). copepod stress Tiedemann, two cold & sustained Hartmann, after Weithoff, reported Dennis, been has the dehydrogonase For NADH (GST). S-transferase in glutathione (20 increase here temperature tested over temperature heat-tolerant up-regulated significantly the were in stress increase (Adachi oxidative sensitivity to heat ultimately related and heat-sensitive Genes stress cellular 2009). increasing accelerates P al., to leading et Puntarulo, temperatures thereby damage elevated (Heise, stress, heat cellular formation to under P to activity ROS organisms contributes Sommer, increases ectothermic and Keller, potentially acids of 2003; and nucleic Exposure respiration and (ROS) 2004). proteins mitochondrial species oxygen Whiteman, modifying reactive called & by are that (Halliwell response compounds cellular toxic of cause production metabolism. which the carbohydrate to and species related is the metabolism, stress between lipid Oxidative differences phosphorylation, significant oxidative expression were in same there involved accelerated the However, genes general, is induction. in in temperature rate had, with its pathways expression and metabolic ased heat-tolerant core temperature the to environmental between belonging to pattern Genes linked increase. inextricably temperature is by ectotherms in Metabolism heat-sensitive and heat-tolerant between cies particular response a for temperature stress Metabolic temperature 4.3 constitutes that condition any under induced are genes in induced were genes hsp40 in also reported hsp40 and and Bcly rcy oeo 07 n ntePcfi oyster, Pacific the in and 2007) Somero, & Gracey, (Buckley, hsp60 hsp70 .fernandoi. B. .manjavacas B. onigtwrsasnritcmcaimrgltn h xrsino hs he ee as genes three these of expression the regulating mechanism synergistic a towards pointing rtis as proteins, .manjavacas B. .calyciflorus B. rnr bl,20) O omto lcsha hc epneadrefolding and response shock heat blocks formation ROS 2004). Abele, & ¨ ortner, o ) mn h ieetal xrse ee eeND eyrgns and dehydrogenase NADH were genes expressed differentially the Among C). ncnrs,teegnswr infiatydw-euae vrtemperature over down-regulated significantly were genes these contrast, In .calysiflorus B. hsp40 .calyciflorus B. Sihe l,21) uhasnritcrltosi a enrpre among reported been has relationship synergistic a Such 2012). al., et (Smith .calyciflorus B. Tigriopus euae h Taeatvt of activity ATPase the regulates .. eeu-euae oad h oe moe eprtr here temperature imposed lower the towards up-regulated were s.s., ihicesn et(mt ta. 02.Ti niae htthese that indicates This 2012). al., et (Smith heat increasing with .calyciflorus B. .fernandoi B. .calyciflorus B. .. ee xdtv tesrsos a nue ttelowest the at induced was response stress oxidative Here, s.s.. o h species the For . ..addw-euae nheat-sensitive in down-regulated and s.s. ..adteha sensitive heat the and s.s. 10 paetysusdw otymtblcprocesses metabolic costly down shuts apparently ..hsaatdt ananismtbls under metabolism its maintain to adapted has s.s. .. infiatidcin(xfl hne of change) fold (7x induction significant a s.s., .japonicas, T. ° Hn en,Ben e,21) In 2018), Lee, & Byeon, Jeong, (Han, C hsp70 rsote gigas Crassostrea ° Brachionus o 0dy (Paraskevopoulou, days 30 for C .fernandoi, B. Cnrn&Tf,20) These 2006). Toft, & (Cintron S ee eesignificantly were genes GST .fernandoi B. Brachionus species Brachionus fe ho acute of 6h after , hwn nincre- an showing rnr Abele, & ¨ ortner, Gillichthys species. Up- . spe- Posted on Authorea 19 Feb 2020 — CC BY 4.0 — https://doi.org/10.22541/au.158212862.25725074 — This a preprint and has not been peer reviewed. Data may be preliminary. aebe ne aoaoycliainoe ogpro ftm n trleso eesv lee(Stelzer, allele recessive a In on 2010). reliess it Riss, and & that time clones of Snell, Wiedlroither, in period & Schmidt, phenomenon long common Pourriot a Stelzer, over a 1974; cultivation 2008; is laboratory sexuality (Gilbert, under abandoning however, composition been rare, have food very is and asexuality density Abandoning conditions population Schr environmental photoperiod, unfavorable 1983; as under recombination such promote factors to Schr sexual 1974; one (Gilbert, and growth population heat-sensitive and Brachionus heat-tolerant in response heat of thermal nus ATP. mechanisms with of cope molecular amount to Other large mechanism 4.5 a saving energy consumes an metabolism or protein homeostasis as cellular stress, reflect may biosynthesis further protein However, 26 Th´ebault,of 2007). to & Moraga, up Tanguy, increase (Meistertzheim, proteins temperature ribosomal of replacement or o heat-sensitive xrsinpolsmgtb sda imrest sesseisvleaiiyt niomna conditions be environmental might to vulnerability stress species heat assess under to upregulated their biomarkers Additionally, be changes. species. as two to climate used the and found of be divergence genes might ecological profiles The the expression to sympatry. expression contributing gene in potentially their selection occur regarding of hence targets temperature) considered can (here, historically genes conditions and been related) environmental had profiles meiosis different What – to growth. species adapted for one rentially in optimal species under – regime single (and genes temperature related environ- the the related stress to beyond while metabolism/translation responses rate, expressed upregulated growth were phenotypes’ highest species of with respective temperature mechanisms the the underlying Generally, indicating change. growth, mental population heat-sensitive in and heat-tolerant differences between heat to responses different chionus significantly found we conclusion, In be this might in up-regulated H3K4 were and CONCLUSIONS same that H3K79 the exposure. pathways under heat of processing species high information trimethylation same under Silencing environmental via the 2017). species numerous for activation Kim, a found Transcriptional & with we Park, exposure. that associated Jeon, suppression heat while (Hyun, translation (H3K4, of transcription, chromatin to genes of silencing related conditions activation to be methyltransferase with might related related chromatin associated been of histone are has In H3K79 of H4K20 histone expression. and of up-regulation to H3K4 methylation gene in regulation of regulating Trimethylation resulted post-translational H4K20). in for temperatures H3K79, role machinery high molecular important et to the Kim an posure lack 2017; play Welch, not which Mark do (Gribble, tails, they regulation However, transcriptional known 2016). or epigenetic is al., methylation for It Dnmt3) acetylation. DNA (Dnmt1, and including methylation transferases on mechanisms, histone studies many including genomic/transcriptomic by tails, from achieved histone be to modifications can ability post-translational here transcription the tested on lost range control the has beyond Epigenetic clone temperatures this by temperature. that triggered than are is other explanations reproduction stimuli 23 sexual Possible or above that temperatures. or low exposure reproduction at temperature sexual nor of that high indicating at meiosis, neither genes, to In related reproduction. genes sexual triggered of up-regulation significant in 20 at ) ugsiga nrae rnlto aaiyo rtcino iooa ucintruhteaddition the through function ribosomal of protection a or capacity translation increased an suggesting C), species o ,idctn gi ht20 that again indicating C, pce.Tasrpoi epne eefudt orlt ihdffrne nfins n especially and fitness in differences with correlate to found were responses Transcriptomic species. dr 05.Rtfrseisaecpbeo bnoigete h eulo h sxa phase. asexual the or sexual the either abandoning of capable are species Rotifer 2005). ¨ oder, sms ooootrtfr,hv w erdciemds n sxa loigfrfast for allowing asexual one modes, reproductive two have rotifers, monogonont most as , .fernandoi B. .calyciflorus B. dr 05.Tesxa hs frpouto sgnrlyidcddet environmental to due induced generally is reproduction of phase sexual The 2005). ¨ oder, iooa rti eae ee eeu-euae ne idha tes(23 stress heat mild under up-regulated were genes related protein ribosomal , o ,rsle ndw-euaino iooa eae ee.Ti suppression This genes. related ribosomal of down-regulation in resulted C, .calyciflorus B. culycmrsssvrlcoeyrltdrtfrseis hc r diffe- are which species, rotifer related closely several comprises actually o ieycmrs tesu odtosfrti pce.I otat nthe in contrast, In species. this for conditions stressful comprise likely C .manjavacas B. .. hr a osgicn prglto fmeiosis-related of up-regulation significant no was there s.s., 11 n te oiesta oieslc N methyl- DNA lack rotifers that rotifers other and .fernandoi B. B nraeo eprtr resulted temperature of increase , . fernandoi efudta ex- that found we , Brachio- Bra- ° C Posted on Authorea 19 Feb 2020 — CC BY 4.0 — https://doi.org/10.22541/au.158212862.25725074 — This a preprint and has not been peer reviewed. Data may be preliminary. ms .&Kly .(05.Otoidr ovn udmna issi hl eoecomparisons genome whole in biases fundamental Solving OrthoFinder: accuracy. 0721-2 learn (2015). inference we orthogroup S. can improves What Kelly, dramatically change: & global to D. responses Emms, evolved and Plastic (2016). transcriptomics? W. comparative M. from Kelly, & B., M. DeBiasse, aquatic changes Publishers. in environmental (Ed), Science tool of Colombus Nova biomarker detection F. as NY: early In proteins York, the shock risks. for heat developments of ecological Expression ongoing and and (2008). knowledge A. Thiery, actual & invertebrates: X., Moreau, chaperone L., Jong, molecular review. De 90-kDa comprehensive The A (1998). applications. G. 79 clinical Nardai, tics, and & Z., function, Prohaszka, structure, C., family: Soti, T., Schnaider, for P., requirements Csermely, the Defining performance. (2006). cardiac D. by Toft, pathway. Indicated & S., responses Abalones transcriptomic N. Pacific Different Cintron, heat-sensitive (2019). (2009). W. You, and T. & heat-tolerant Physiology C., Madden, Ke, in in X., Frontiers & Luo, stress K., Y., thermal Shen, Bealer, C., to Lu, J., Z., goby Papadopoulos, Huang, N., the N., Chen, in Ma, stress applications. V., heat and Avagyan, Architecture to G., BLAST+: response Coulouris, cellular C., The Camacho, jeb.02292 (2006). sequence 10.1242/ N. Illumina doi: G. for 2660–2677. Somero, trimmer , & flexible Y., A mirabilis A. Gillichthys Trimmomatic: Gracey, A., (2011). B. B. Buckley, Usadel, & M., Lohse, data. M., A. the V. Pavlichenko, of Bolger, G., organization D. and ( Baikal. patterns Garbuz, species Lake Expression V., from amphipod (2013). from M. endemic ) G. Protopopova, congener Retrieved O. two A., data. in Zatsepina, M. thermotolerance & sequence Timofeyev, , . B., throughput . . M. high V., Evgen’ev, for S., D. tool Bedulina, control quality a Oxidative recovery. FastQC: (2009). protein http://www.bioinformatics.babraham.ac.uk/projects/fastqc Y. (2010). unfolded Shinomura, S. & delays Andrews, K., and Imai, response A., Nakai, stress K., journal.pone.0007719 heat S. 10.1371/ the Calderwood, K., impairs Fujii, stress from Y., clusters Liu, gene M., co-expressed Adachi, optimal of extraction Automatic data. Clust: expression (2018). gene S. Kelly, computing & their B., of Abu-Jamous, use the for Ende growth Marco Dr. population REFERENCES and the Lenhard of Michael analysis Dr. bootstrapping Prof. R thank servers. in to additionally like contributing to We we like for experiments. We Wacker Finally life-table change”. Alexander data. the climate Dr. performing and in thank “Adaptation helping to area for want Schirmer focus Christina Potsdam, technician of the University thank by supported was work This ACKNOWLEDGEMENTS iifrais 30 Bioinformatics, 2,1918 o:10.1016/s0163-7258(98)00013-8 doi: 129–168. (2), h ora fBooia hmsr,281 Chemistry, Biological of Journal The oeua clg,22 Ecology, Molecular eoeBiology Genome DAmcora n rti-ee analysis, protein-level and microarray cDNA a : , 9 1422.di 10.1093/bioinformatics/btu170 doi: 2114–2120. , 85 o:1.39 fphys.2018.01895 10.3389/ doi: 1895. , ora fHeredity of Journal , 19 M Bioinformatics BMC 4613.di 10.1111/mec.12136 doi: 1416-1430. , 7.di 10.1186/s13059-018-1536-8 doi: 172. , etsokpoen:ItrainlResearch International proteins: shock Heat 63–64.di 10.1074/jbc.M605417200 doi: 26235–26244. , 12 eoeBooy 16 Biology, Genome , 107 18.doi:10.1093/jhered/esv073 71–81. , uingmau cyaneus Eulimnogammarus , Hsp40 10, 2.di 10.1186/1471-2105-10-421. doi: 421. ora fEprmna Biology Experimental of Journal and Hsp70 5.doi:10.1186/s13059-015- 157. , hraooy&Therapeu- & Pharmacology LSone PLoS hsp70 nthe in ee orlt with correlate genes p.7-9) New (pp.375-392). and Hsp90 , 4 .verrucosus E. 71.doi: e7719. , chaperone , 209 Posted on Authorea 19 Feb 2020 — CC BY 4.0 — https://doi.org/10.22541/au.158212862.25725074 — This a preprint and has not been peer reviewed. Data may be preliminary. eran . aida,S . cwn,K,&Crele,M 21) ouaintasrpoisin transcriptomics Population (2018). M. Cordellier, & K., Schwenk, P., S. Daphnia Ravindran, M., Pharmacology Herrmann, & Toxicology C: Part isolated by - species 0456(02)00212-0 Physiology oxygen reactive and of Production Biochemistry bivalve tive (2003). Antarctic D. Abele, the & of O., mitochondria H. Portner, S., Puntarulo, K., Heise, copepods reac- congeneric of two generation in the on transferases changes glutathione-S temperature 018-1224-3 of of activity Effects and (2018). japonicus expression in S. Tigriopus the J. and and Lee, & species vivo E. oxygen mean? in Byeon, tive B., results damage C. the Jeong, oxidative do J., and Han, what species and it reactive do Measuring doi:10.1038/sj.bjp.0705776 you 231–255. should (2004). How (Eds), M. Fath culture: Whiteman, cell B. & & B., Jorgensen E. Halliwell, S. In Elsevier. UK: Range. London, Tolerance 3557–3564), Chlorophyllide. (2008). with J. A.J. algae Hanna, Yellow-green Cullum, & (1972). J. P., C. S. Lorenzen, Gygi, & , V, L., Response R. V. Stress R. Eapen, Proteotoxic Guillard, Effective P., an D. Chemistry as Waterman, Biological Reduction of K., Ribosome Journal Identifies M. Analysis Bhanu, Proteomic M., (2015). rotifer Isasa, the in A., aging (2011). Guerra-moreno, of transcriptomics A. Genome-wide Regev, (2017). B. . D. . Welch, . manjavacas Mark I., & Amit, E., A., K. Gribble, D. genome. Thompson, reference Z., a J. without Levin, data 29 M., RNA-Seq Yassour, from assembly J., transcriptome B. Full-length Haas, G., M. complexes species Grabherr, cryptic ancient of in Speciation phylogeny 10.1111/j.0014-3820.2002.tb01455.x molecular (2002). H. the doi: D. from Lunt, & evidence R., G. : Carvalho, M., Serra, A., Gomez, Rotifers. in 10.1038/NCLIMATE2810 Dormancy doi: (1974). 490–513. J. ecological and J. similarity Morphological Gilbert, (2013). species. J. M. rotifer Carmona, two & in M., overlap Serra, J., the Montero-Pau, from C., Gabaldon, learn can we differentiation 10.1007/s10750-016-2723-9 and Ecological what doi: taxon- Phylogenetics (2017). M. 7–18. species: DNA Molecular Serra, & rotifer by J., cryptic Montero-Pau, revealed (2009). J., in M. (Rotifera) Carmona, G. D., animals Fontaneto, T. C., microscopic Gabaldon, Barraclough, in & diversity A., hidden of E. omy. levels Herniou, Extreme M., Evolution two Kaya, in D., stasis morphological Fontaneto, the Disentangling (2007). M. Serra, 007-0573-1 & the G., of species Melone. rotifer I., Giordani, D., response: Fontaneto, stress the and chaperones, molecular proteins, physiology nurev.physiol.61.1.243 Heat-shock ecological (1999). and E. G. evolutionary Hofmann, & E., M. Feder, 8 4–5.di 10.1038/nbt.1883 doi: 644–652. , 10–14. , oeua hlgntc n Evolution and Phylogenetics Molecular h oeo hra selection. thermal of role The : neegn oe system. model emerging an , rcinsplicatilis Brachionus and irou kingsejongensis Tigriopus LSone PLoS , 290 aenl elliptica Laternula 5) 99–90.di 10.1074/jbc.M115.684969 doi: 29695–29706. (50), nulRve fPyilg.61 Physiology. of Review Annual . :507 doi:10.1371/journal.pone.0057087 8:e57087. . pce complex species oeua clg,27 Ecology, Molecular M Genomics BMC , rcinsplicatilis Brachionus 53 rnatoso h mrcnMcocpclSociety Microscopical American the of Transactions 1,1219 o:10.1016/j.ympev.2009.04.011 doi: 182–189. (1), . 13 ihre Science Fisheries rcinsplicatilis Brachionus yrbooi,583 Hydrobiologia, Kn n rdrp ne etstress. heat under Broderip) and (King , 18 1.di 10.1186/s12864-017-3540-x doi: 217. , 8–0.di 10.1111/mec.14450 doi: 387–402. , (Rotifera), rts ora fPharmacology of Journal British , 84 , 134 9–0.di 10.1007/s10750- doi: 297–307. , 1–2.doi:10.1007/s12562- 815–823. , vlto,56 Evolution, 4–8.di 10.1146/an- doi: 243–282. , complex. nylpdao ecology of Encyclopedia 99.doi:10.1016/S1532- 79–90. , aueBiotechnology, Nature ora fPhycology of Journal yrbooi,796 Hydrobiologia, 7,1431–1444. (7), Brachionus Compara- , , 142 93 (pp. The , , , Posted on Authorea 19 Feb 2020 — CC BY 4.0 — https://doi.org/10.22541/au.158212862.25725074 — This a preprint and has not been peer reviewed. Data may be preliminary. a . iY . hn,J . e,X . in,X .(00.Dffrne nlf al demography table life in Differences (2010). L. X. Xiang, & L., X. Wen, of Y., populations J. geographic Zhang, eight L., gene applicable Y. among generally Xi GAGE: Q., (2009). P. Ma, Woolf, & analysis. toler- pathway K., salinity Hankenson, for enrichment K., does set Shedden, integration M., How data Friedman, pathway-based W., for Luo, package (2007). R/Bioconductor an S. “Pathview: J. visualization.” (2013). D. and C. Brouwer, Montagnes, & W. & Luo, C., Diaz-Avalos, of J., distributions doi:10.1007/s00227-006-0366-5 S. the influence Kemp, RNA-seq ance for D., dispersion and C. change fold Lowe, of estimation Moderated (2014). DESeq2. S. with Anders, & data W, aggregates. Huber, material I., M. of Love, growth stress of Thermal mode (2016). the S. on 199–216. J. Studies , Rhee, & 1907. Proteomics J., J., Y. and A. oyster Kim, Lotka, Genomics Pacific Y., D: I. the Part Choi, in S., - J. profile Physiology rotifer Lee, transcriptome and J., the distinct I. of Hwang, a M., COI induces B. by Kim, identified J., H. succession Lim, temporal Rapid (2010). 32 R. Research, Ma, & C., calyciflorus without genome or Niu, The with L., (2018). data RNA-seq S. Li, from J. quantification Lee, transcript accurate genome. . RSEM: . reference . (2011). a C., C.N. Dewey, M. & Lee, B., S., Li, Correlation D. Hwang, (2016). B., C. S. 10.1111/1755-0998.12768 Jeong, rotifer J. doi: J., monogonont Lee, Han, freshwater Y., & the B. of S., Lee, J. S., H. Rhee, oyster, Kim, J., rotifer, Pacific in Chipman, the (5mC) 5-methylcytosine fish. Kevin of genome-wide and and A., response family copepod, gene Mally, Transcriptome (Dnmt) methyltransferase L., DNA (2017). the Mirbahai, between S. M., J. B. Rhee, Kim, & Y., Toxicology I. Cellular & Choi, analysis. K., data Kim, RNA-seq gigas Crassostrea of M., out B. most and the Kim, Getting functioning (2015). energetic Y. of C. Lau, Seasonality 10.7717/peerj.1360. & F., (2004). of T. D. Khang, cost Abele, and ( O., parameters lugworm by H. changes. history species perature Portner, oxygen life M., reactive on of A. production temperature Sommer, of M., effect Keller, The methylations. (2011). rotifer lysine E. 10.1080/02705060.2011.563998 histone the H. reading in and Enesco, reproduction erasing & Writing, P., (2017). Kauler, J. Kim, 49 & Medicine, Molecular K., & Park, In Experimental ecology. J., insect Jeon, Aquatic K., (2010). Hyun, M. Elsevier. (Eds), R. UK: Covich Northington, London, P. & 659–694), A. T., (pp. & D. Thorp, Chaloner, J. A., G. H. Lamberti, E., A. Hershey, alsi ia od,Biig,Cia,i eaint clgcltraits. ecological to relation in , China , Beijing , Pond Xihai in Pallas 5-5.di 10.1093/plankt/fbq014 doi: 951-959. , ucpil otemlsrs:Acmaio ihtersos ftlrn oyster. tolerant of response the with comparison A stress: thermal to susceptible ora fEprmna ilg,207, Biology, Experimental of Journal iifrais 29 Bioinformatics, ee eois 38 Genomics, Genes eoeBiology Genome M iifrais 12 Bioinformatics, BMC , 13 0–1.doi:10.1007/s13273-017-0011-z 105–113. , rcinscalyciflorus Brachionus rcinsplicatilis Brachionus clg n lsicto fNrhAeia rswtrinvertebrates, freshwater American North of classification and Ecology M Bioinformatics BMC rcinscalyciflorus Brachionus , ,13–81 o:10.1093/bioinformatics/btt285 doi: 1830–1831. ), 15 rcinscalyciflorus Brachionus 34 o:013/emm.2017.11 doi:10.1038/ e324. , 32.di 10.1007/s13258-015-0333-y doi: 13–23. , 5.di 10.1186/s13059-014-0550-8 doi: 550. , 2.di 10.1186/1471-2105-12-323. doi: 323. : rncl marina Arenicola , 14 , ora fFehae clg,26 Ecology, Freshwater of Journal 19 5923.di 10.1242/jeb.01050 doi: 2529–2538. iln species? sibling 27.di 10.1016/j.cbd.2016.06.006 doi: 62–70. , , 10 . rsote gigas Crassostrea 6.doi:10.1186/1471-2105-10-161 161. , oeua clg eore,18 Resources, Ecology Molecular Rtfr)fo China. from (Rotifera) iohnraepsdt ct tem- acute to exposed mitochondria ) mrcnJunlo cec 141 Science of Journal American aieBiology Marine . oprtv Biochemistry Comparative er,3 PeerJ, ora fPlankton of Journal Limnologica 9–0.doi: 399–408. , , 150 16.doi: e1360. : Brachionus 377–386. , 646–655. , Molecular , 40 , Posted on Authorea 19 Feb 2020 — CC BY 4.0 — https://doi.org/10.22541/au.158212862.25725074 — This a preprint and has not been peer reviewed. Data may be preliminary. orit . nl,T .(93.Rsigeg nrotifers. in eggs Resting (1983). W. T. Snell, & to doi:10.1007/BF00045970 acclimation R., with associated Pourriot, expression (1974). gene killifish W. in annual R. Poole, an Changes in 207 Biology, temperatures (2004). Experimental daily N. of degrada- fluctuating Journal G. and tolerance: Somero, temperatures stress & constant in E., proteins J. heat-shock Podrabsky, of function proteins. The damaged (1993). of nurev.ge.27.120193.002253 S. reactivation Lindquist, and change. & tion climate A., recent D. to Parsell, among responses 37 evolutionary Systematics, stress and and heat Ecological Evolution, Ecology, to (2006). response C. complex. Differential Parmesan, species invertebrate (2018). aquatic G. an 10.1098/rsbl.2018.0498. Weithoff, of R., lineages evolutionary Tiedemann, species Within S., rotifer the (2019). Paraskevopoulou, in R. temperatures Tiedemann, different & to response S., of expression Hartmann, calyciflorus gene structure G., and Weithoff, variability population genetic B., sculpturing expressed A. Dennis, processes S., microevolutionary Paraskevopoulou, Allochronic two (2013). J. T. : Abatzopoulos. succession & Brachionus I., clonal Kappas, (2016). and A., S.A.J. Triantafyllidis, Declerck, divergence E., Michaloudi, evidence . N., . . discordance: S. Papakostas, J., mitonuclear complex. Rota, species widespread cryptic L., despite rotifer Verhage, units a M., evolutionary from M. Brehm, Thomas recognizes K., & taxonomy Proios, C., Integrative E., C. Michaloudi, Murdock, S., S., Papakostas, change. Blanford, 23630036 climate I., PMID: to doi:10.1111/gcb.12240 J. sensitive 2373–2380. Blanford, more 19, A., ectotherms , R. makes an- variation Seliga, genome Temperature L., automatic (2013). R. B. An Heinig, KAAS: P., (2007). K. M. Paaijmans, Kanehisa, C., server. A. reconstruction Yoshizawa, pathway S., and rotifer Okuda, notation of M., Itoh, coexistence Y., Long-Term Moriya, (2011). G´omez, A. & M. species, cryptic Serra, Ramos-Rodr´ıguez, E., J., Montero-Pau, the deciphering Walsh, taxonomy. one: DNA in . . through . species gononta) H., Fifteen K. (2017). Galindo, J. A., E. Hagiwara, G´omez, Alc´antara-Rodr´ıguez, Ciros-P´erez, A., J., S., A., Mills, J. description (re) J. the allows A. and species. analysis S. phylogenetic four molecular Declerck, of by revealed & delimitations W, species Tihlaˇrıkov´a, confirms Zhang, the Nedˇela,lysis V., to E., G., applied differentially taxonomy Stamou, Reverse of (2018). S., Identification Papakostas, E., (2007). Michaloudi, T. M. oyster Thebault, Pacific & the D., Journal of Moraga, genes A., Tanguy, expressed L., A. Meistertzheim, 62, Sciences, Life Molecular (2005). and B. Bukau, & M.P., Mayer, j.limno.2009.05.002 10.1016/ doi: 16–22. 7,69–42 doi:10.1111/j.1742-4658.2007.06156.x 6392–6402. 274, , rotifers, es stricto. sensu LSoe 13 one, PLoS LSo,6 on, PLoS yrbooi,700 Hydrobiologia, nrdcint uniaieEcology Quantitative to Introduction LSone PLoS 250 o:10.1371/journal.pone.0021530.t001 doi: e21530. , 0018 o:10.1371/journal.pone.0203168 doi: e0203168. , 7-4 o:10.1007/s00018-004-4464-6 doi: 670-84. Hydrobiologia Hsp70 4 0214 doi:10.1371/journal.pone.0223134 e0223134. 14, , rsote gigas Crassostrea ytmtcBooy 65 Biology, Systematic 2725.di 10.1242/jeb.01016 doi: 2237–2254. , rcinscalyciflorus Brachionus 34.di 10.1007/s10750-012-1217-7 doi: 33–45. , 3–6.di1.16 annurev.ecolsys.37.091305.110100 doi:10.1146/ 637–669. , hprns ellrfntosadmlclrmechanism. molecular and functions cellular chaperones: uli cd eerh 35 Research, Acids Nucleic nulRve f eeis 27, Genetics, of. Review Annual , 796, 15 rcinsplicatilis Brachionus 95.doi:10.1007/s10750-016-2725-7 39–58. xoe opoogdtemlstress. thermal prolonged to exposed e ok Y McGraw-Hill NY: York, New . 0–2.di 10.1093/sysbio/syw016 doi: 508–524. , rpi pce ope:Mrhmti ana- Morphometric complex: species cryptic ilg etr,14 Letters, Biology 8–8.di 10.1093/nar/gkm321 doi: 182–185. , pce ope Rtfr,Mono- (Rotifera, complex species yrbooi.104, Hydrobiologia. 3–9 o:10.1146/an- doi: 437–49. utoudlslimnaeus. Austrofundulus lblCag Biology Change Global nulRve of Review Annual 0848 doi: 20180498. , Brachionus h FEBS The 213–224. Cellular Posted on Authorea 19 Feb 2020 — CC BY 4.0 — https://doi.org/10.22541/au.158212862.25725074 — This a preprint and has not been peer reviewed. Data may be preliminary. tle,C .(07.Lf itr aito nmngnn oies nA aiaa&T ohng (Eds), Yoshinaga T. & Hagiwara A. In rotifers. monogonont in in dwarfing variation and history reproduction Life sexual Rotifers (2017). of P. Loss signals C. (2010). Stelzer, sexual S. of Riss, & role metazoan. A., the small Wiedlroither, and a J., Schmidt, rotifer P., a C. Stelzer, in asex Obligate (2008). Biology P. transcript-level C. histories. Stelzer, : life RNA-seq rotifer for of analyses reproduction. Evolution Differential of 005-4243-x (2005). cost P. (2016). C. the D. Stelzer, M. rotifers: Robinson, in inferences. & gene-level patterns I., improve fecundity M. estimates and Love, Lifespan C., Soneson, (1977). Hoarau E. & es- C. O., species, 31 M. zooplankton King, are Evolution, J. key W., proteins Fernandes, two T. M., in shock Y. Snell, stress heat A. thermal Three Sundaram, to P., response Lindeque, transcriptome (2012). finmarchicus F., Contrasting E. W. (2015). Moller, T. G. S., Snell, Kollias, I., & Smolina, T., Shearer, R., thermotolerance. 10.1016/j.jembe.2011.11.027. A. rotifer Burns, for sential A., H. Smith, the in (Eds), Speciation Yoshinaga copepod T. (2017). intertidal & D. The Fontaneto, & (2006). M. S. Serra, J. 340 Lee, Communications. & Research G., Biophysical ( H. and gene freshwater. Park, 20 M., protein in shock Y. heat (Rotifera) Lee, S., rotifers J. of Seo, diversity Global doi:10.1007/s10750-007-9003-7 (2008). H. Segers, Rotifers. Monogonont in molecular Diapause the 005-4235-x Investigating (2005). transcriptome (2012). T. the S. across Schroder, expression R. gene Burton, in & differences Population A., copepod Wolff, stress: the controlling W., thermal of to G. mechanism adaptation Moy, local the S., of F. basis with Barreto, associated D., S. changes Schoville, rotifer. Biochemical aging the (1990). evolution in C. 10.1002/jcb.240440304 the formation J. and radical expression Carlson, gene superoxide & modula- of studies M., biochemical Comparative Sawada, and (2012). Molecular Y. Gilad, regulation. & gene (2011). I., of Ruvinsky, J. G., Lee, I. Romero, & Y., Lee, J., ( 10.1016/j.cbpc.2011.02.009 Lee, 20 protein rotifer H., shock monogonont Choi, heat R., of Statistical tion Kim, for Foundation J., R computing. Rhee, statistical for (http//:www.R-projest.org). environment Austria. and language Vienna, A Computing, R: (2013). CoreTeam R , p.8–0) igpr:Springer. Singapore: 88–109), (pp. , 21 8–9.doi:10.1111/j.1420-9101.2007.01437.x 287–293. , and 8–9.di 10.1111/j.1558-5646.1977.tb01082.x doi: 882–890. , irou aioncs M vlto ilg,12 Biology, Evolution BMC californicus. Tigriopus .glacialis C. LSone PLoS Brachionus aueRve eeis 13 Genetics, Review Nature Rotifers Hsp20 , . 5 p.53) igpr:Springer. Singapore: (pp.15–32), , Hsp20 sp. aieEooyPors eis 534 Series, Progress Ecology Marine 184 o:10.1371/journal.pone.0012854 doi: e12854. , nacstemtlrneo transformed of thermotolerance enhances ) ora fEprmna aieBooyadEooy 413 Ecology, and Biology Marine Experimental of Journal oprtv iceityadPyilg,Pr C Part Physiology, and Biochemistry Comparative eeb eprtr tesadhdoe eoie(H peroxide hydrogen and stress temperature by gene ) 10Rsac,4, F1000Research, 0–1.di 10.1038/nrg3229 doi: 505–516. , 0–0.di 10.1016/j.bbrc.2005.12.086 doi: 901–908. , ora fClua iceity 44 Biochemistry, Cellular of Journal rcinu plicatilis Brachionous 16 yrbooi,546 Hydrobiologia, yrbooi,546, Hydrobiologia, 51 o:10.12688/f1000research.7563.1 doi: 1521. 1,10 o:10.1186/1471-2148-12-170. doi: 170. (1), 99.di 10.3354/meps11398 doi: 79–93. , pce ope.I .Hagiwara A. In complex. species 3–4.di 10.1007/s10750- doi: 335–346. , Hydrobiologia shrci coli Escherichia 9–0.doi:10.1007/s10750- 291–306. ora fEvolutionary of Journal . irou japonicus Tigriopus , 154 5–6.doi: 153–165. , , . 595 92.doi: 19–27. , Biochemical 2 –.doi: 1–6. , O 2 49–59. , Calanus nthe in ) small Posted on Authorea 19 Feb 2020 — CC BY 4.0 — https://doi.org/10.22541/au.158212862.25725074 — This a preprint and has not been peer reviewed. Data may be preliminary. in,X,X,Y . hn,J . a . e,X 21) ffcso eprtr nsria,repro- survival, on temperature of Effects the (2010). X. in Wen, processes & two and Q., of Patterns Ma, offspring 25 Y., Ecology, in (2011). Freshwater J. morphotype Zhang, K. and L., Hu, duction, Y. & Xi, X., X., J. Xiang, Wang, Evolution, G., and Zhang, Phylogenetics Molecular X., among the characteristics Wen, of history differentiation Y., life genetic Xi, in X., Differences Xiang, (2016). Y. Xi, the & doi:10.1080/02705060.2016.1141379 of Y., 351–360. species Chen, evolutionary Y., sympatric Tao, three R., differen- Jiang, genetic population X., and LimnologieXiang, phylogeny Molecular A. de Annales (2017). L. L. Limnology: Romano, Y. of Ge, & Journal & V., L, in X. M. Wen, patterns L., Rockman, tiation Y. M., Xi, L., Pizer, X. P., Xiang, eukaryotes. J. in Balhoff, regulation transcriptional E., doi:10.1093/molbev/msg140 of Abouheif, 1377-1419. evolution W., The M. (2003). Hahn, A., G. Wray, cryptic of Coexistence (2016). H. (2016). Wickham, X. variables, Xiang, environmental & of Y., roles on 10.1093/plankt/fbw006 Xue, deprivation G., food species: Zhang, of (Rotifera) length Y., the Xi, of X., effect Wen, the – species reproduction. rotifer and two span in life restriction Dietary habitat. (2007) extreme G. an Weithoff, in (Rotatoria) ecological consumers two the of confine separation , niche stressors Vertical environmental (2004) food G. Multiple Weithoff, the (2013) determines G. rotifer flagellates Weithoff, the mixotrophic & of niche N. of nutrition Laufenstein, history of T., life Weisse, mode in The Differences consumers. (2014). (2007). their L. for A. Y. quality Wacker, 50, Xi, & Limnology, & G. of L., Weithoff, Journal Huang, International Y., - Han, Limnologie in N., de species M. Annales sibling Xia, two L., between X. characteristics rotifer Xiang, L., the tem- X. and of Wang, M. performance complex Clark, between species relationships & intraspecific cryptic F., doi:10.1007/s10750-014-1859-8 and O. a Inter- D. in Skibinski, (2014). bivalve perature M. S., Antarctic Serra, the L. & Peck, in Walczy´nska, 391, A., stress G., Ecology, heat Hillyard, and to Biology S., Marine response A. Experimental Transcriptional M. Thorne, (2010). G., S. Burns, reproduction between M., Trade-off Truebano, (2017). Y. Chen, & boundaries Y., 10.1038/s41598-017-15863-y Huang, species rotifer X., doi: of the Zhu, L., analysis of — Zhang, lifespan An X., rotifer and Xu, The X., (2006). Hou, A. : Y., Sun, Caccone, complex & species 41 W., cryptic Evolution, T. a and Snell, in Phylogenetics S., patterns Rice, biogeographic S., and Vicario, E., Suatoni, 139 9-0.di 10.1007/s00442-004-1545-z doi: 594-603. , rcinscalyciflorus Brachionus ehldlaacidophila Cephalodella glt:EeatGahc o aaAnalysis Data for Graphics Elegant ggplot2: –8 o:10.1080/02705060.2010.9664352 doi: 9–18. , rcinsplicatilis Brachionus Oecologia ucinlEcology Functional rcinscalyciflorus Brachionus 69.di 10.1016/j.ympev.2006.04.025 doi: 86–98. , , 153 9 8–9.di 10.1016/j.ympev.2011.02.011 doi: 386–398. 59, als(oiea ope rmtolksi China. in lakes two from complex (Rotifera) Pallas , 0-0.di 10.1007/s00442-007-0739-6 doi: 303-308. , oai rotatoria Rotaria rswtrBiology Freshwater rcinscalyciflorus Brachionus rcinscalyciflorus Brachionus , ne ieetfo conditions. food different under , 53 57.di1.06 j.jembe.2010.06.011 doi:10.1016/ 65–72. 21 0–1.di 10.1051/limn/2017024 doi: 401–410. , ope,apsieydsesn rswtrzooplankton. freshwater dispersing passively a complex, 0219.di 10.1111/j.1365-2435.2007.01333.x doi: 1092–1098. , 17 ora fPako eerh 38 Research, Plankton of Journal rcinsplicatilis Brachionus complex. , 58 8–9.di 015/in/2014024 10.1051/limn doi: 289–298. 2de.,NwYr,N:Springer. NY: York, New ed.), (2nd oeua ilg n Evolution and Biology Molecular 0811.di 10.1111/fwb.12104 doi: 1008-1015. , ope rmtoia hlo lakes. shallow tropical from complex Rtfr)morphotypes. (Rotifera) ora fFehae clg,31, Ecology, Freshwater of Journal rcinsplicatilis Brachionus . aenl elliptica Laternula Hydrobiologia cetfi eot,7 Reports, Scientific rcinscalyciflorus Brachionus 7–8.doi: 478–489. , , 734 International . ora of Journal ora of Journal . Molecular Oecologia 17–26. , 15370. , , 20 , Posted on Authorea 19 Feb 2020 — CC BY 4.0 — https://doi.org/10.22541/au.158212862.25725074 — This a preprint and has not been peer reviewed. Data may be preliminary. ihcnrbtosfo l uhr.Alcnrbtn uhr edadare otefia eso fthe of version final the the of to statistics agreed S.P,and Summary and by read coordinated 1: authors was TABLE contributing finalization R.T.. All from manuscript input and with authors. editing A.B.D, all manuscript. and Further S.P. from S.P. by contributions by out performed carried were with drafted was experiments was life-table results the of manuscript and interpretation work The and lab The analysis G.W. Data and S.P. S.P, by R.T. by repository. conceived other was Dryad All project on The deposited SRR10426055-76). be numbers: will (accession study CONTRIBUTIONS database this AUTHOR Bank of findings Gen the the support from that available data are (2009). files M. SRA G. All Smith, & patterns A., Temporal A. ACCESSIBILITY Saveliev, (2017). DATA J., L. R N. X. Walker, with Xiang, ecology N., in E. . extensions Leno, . . F., P., A. Xie, Zuur, F., L. the Ning, of Q., differentiation lake. Sun, shallow genetic L., of Y. processes the Xi, differentiation and of of A., Patterns Zhou, species (2019). Y., described J. Zhang, recently A. four S. of Declerck demography & complex. and S., species themes history Papakostas biological L., life comparing Zhou the for Pacific D., in package K. the R Lemmen, an of W., ClusterProfiler: Zhang, response (2012). transcriptional Q. He, The & clusters. gene Y., among (2017). Han, L., Z. Wang, G., Zhou, Yu, & L., Wang, C., 10.1016/j.fsi.2017.07.016 Liu, Q., oyster Gao, C., Yang, .fernandoi B. rsote gigas Crassostrea Otrs5976,477 1,490 457 5,947 1,827 854.87 19,440 30.31 160,070,295 526 94,884 1013.05 224,735 130,682,421 430,046,346 17,973 28.19 798,693,466 72,165 144,037 (%) terms assembly KO entire # 187.245 the for content GC (bp) N50 (bp) bases assembled Total 358,527,278 (bp) length Median (bp) length Average (n) 128,999 ORFs predicted # (n) “unigenes” (n) removal assembled 670,242,336 contamination # after contigs assembled (n) # assembled (n) reads contigs raw assembled quality # high and Trimmed # (n) reads Raw # statistics assembly Transcriptome yrbooi,807 Hydrobiologia, r swtrBiology Freshwater MC:AJunlo nertv Biology Integrative of Journal A OMICS: transcriptomes. gis ct etstress. heat acute against e ok Y Springer. NY: York, New . 1–3.di 10.1007/s10750-017-3407-9 doi: 313–331. , , 64 9420.doi:10.1111/fwb.13388 1994–2005. , enovo de rcinscalyciflorus Brachionus 18 ihadSels Immunology Shellfish and Fish sebiso the of assemblies , 16 .calyciflorus B. 5,2427 o:10.1089/omi.2011.0118 doi: 284–287. (5), Rtfr)cmlxi subtropical a in complex (Rotifera) rcinscalyciflorus Brachionus rcinscalyciflorus Brachionus s.s. ie ffcsmdl and models effects Mixed , .fernandoi B. 68 3–4.doi: 132–143. , cryptic s.s. Posted on Authorea 19 Feb 2020 — CC BY 4.0 — https://doi.org/10.22541/au.158212862.25725074 — This a preprint and has not been peer reviewed. Data may be preliminary. (26/23 heat-sensitive the 1. Figure o )adhg (32/26 high and C) xeietldsg o rncitmcrsosst etepsr ewe ettlrn and heat-tolerant between exposure heat to responses transcriptomic for design Experimental Brachionous o )ha o ahspecies. each for heat C) pce.Tmeauergmswr hsnt ersn oto (20 control represent to chosen were regimes Temperature species. 19 o ) mild C), Posted on Authorea 19 Feb 2020 — CC BY 4.0 — https://doi.org/10.22541/au.158212862.25725074 — This a preprint and has not been peer reviewed. Data may be preliminary. otta rcdr ih19iterations. 199 with procedure bootstrap of responses of responses denote lines Solid calyciflorus 2. Figure ..(cl n h heat-sensitive the and (Bcal) s.s. alnMirsria uvs() n ouaingot rate/ growth Population and (A), curves survival Kaplan-Meier .calyciflorus B. . 2,2,2,3) oe eoete9%cndneitrasetmtdvaa via estimated intervals confidence 95% the denote Boxes 32). 26, 23, (20, s.s .fernandoi B. odffrn eprtrs(0 3 6,wiedse ie represent lines dashed while 26), 23, (20, temperatures different to .fernandoi B. 20 Be)udrdffrn eprtr conditions. temperature different under (Bfer) r B o h heat-tolerant the for (B) B. Posted on Authorea 19 Feb 2020 — CC BY 4.0 — https://doi.org/10.22541/au.158212862.25725074 — This a preprint and has not been peer reviewed. Data may be preliminary. ramn.Tesmosidct h ariecmaio nwihdffrnilgn xrsin(E was (DE) expression gene differential heat which high in represents (H) comparison color pairwise red the and indicate treatment, heat symbols mild The represents (M) treatment. color orange treatment, control heat-tolerant 4. Figure one. lower the regulation over down- temperature and higher Up- the treatment. of heat high perspective represents the (H) from color defined red is and treatment, heat-tolerant heat the mild for represents (FDR=0.05) heat-sensitive analyses the DESeq2 the and by determine as comparisons 3. Figure endarm hwn h ubro ieetal xrse ee npiws temperature pairwise in genes expressed differentially of number the showing diagrams Venn etmpuignraie onswihso atrso xrsinin expression of patterns show which counts normalized using map Heat .calyciflorus B. .fernandoi B. ..() n h heat-sensitive the and (A), s.s. B.Bu oo C ersnscnrltetet rneclr(M) color orange treatment, control represents (C) color Blue (B). 21 .fernandoi B. B.Bu oo C represents (C) color Blue (B). .calyciflorus B. hsp ee o the for genes ..(A) s.s. Posted on Authorea 19 Feb 2020 — CC BY 4.0 — https://doi.org/10.22541/au.158212862.25725074 — This a preprint and has not been peer reviewed. Data may be preliminary. iue6. Figure tolerant, heat the for captured changes heat-sensitive represent side control Left in (B). categories biological KEGG 5. Figure (red). expression (p significant itiuino ieeta xrse D)KG ahas(D=.5 nmi n secondary and main in (FDR=0.05) pathways KEGG (DE) expressed differential of Distribution hrdadcnrsigc-xrsinpten fgn rhgop Os ewe heat-tolerant between (OGs) orthogroups gene of patterns co-expression contrasting and Shared < .fernandoi B. .5.Teclrkyrpeet pcrmo oetgn xrsin(le ohgetgene highest to (blue) expression gene lowest of spectrum a represents key color The 0.05). . vs. idha xoue() n nmild in and (A), exposure heat mild 22 .calyciflorus B. ..wiergtsd o the for side right while s.s. vs. ihha exposure heat high Posted on Authorea 19 Feb 2020 — CC BY 4.0 — https://doi.org/10.22541/au.158212862.25725074 — This a preprint and has not been peer reviewed. Data may be preliminary. rga.Tettlnme n oepeso atr falcutr r ie nFgr S9. Figure in given are clusters all of pattern co-expression and number total The program. heat-sensitive and Brachionus pce.C,C,C ersn h ubr fcutr rdcdby produced clusters of numbers the represent C7 C6, C1, species. 23 Clust