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Sex allocation plasticity on a transcriptome scale Ramm, Steven A; Lengerer, Birgit; Arbore, Roberto; Pjeta, Robert; Wunderer, Julia; Giannakara, Athina; Berezikov, Eugene; Ladurner, Peter; Schärer, Lukas Published in: Molecular Ecology

DOI: 10.1111/mec.15077

IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from it. Please check the document version below. Document Version Final author's version (accepted by publisher, after peer review)

Publication date: 2019

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Citation for published version (APA): Ramm, S. A., Lengerer, B., Arbore, R., Pjeta, R., Wunderer, J., Giannakara, A., Berezikov, E., Ladurner, P., & Schärer, L. (2019). Sex allocation plasticity on a transcriptome scale: socially-sensitive gene expression in a simultaneous hermaphrodite. Molecular Ecology, 28(9), 2321-2341. https://doi.org/10.1111/mec.15077

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Download date: 07-10-2021 Accepted Article 1 Athina Giannakara Athina 2 4 3 Steven A. Ramm Running title: Sex allocationplasticity in hermaphrodites hermaphrodite simultaneous a in expression gene socially-sensitive scale: atranscriptome on plasticity allocation Sex Article Article type : Original 0000-0001-7786-7364) : ID (Orcid RAMM A STEVE DR. This article by article rightsprotected This reserved. is All copyright. 10.1111/mec.15077 doi: lead to differences between this version the Versionand of Record. Please cite this article as pa typesetting, copyediting, through the been publication for accepted hasbeen article This ‡ Current‡ address: Institutdes Biosciences,Université de Mons, Belgium Corresponding† author(email: [email protected]) Evolutionary Biology, Bielefeld University, Germany University, Bielefeld Biology, Evolutionary Institute of Zoology &CMBI, University of Innsbruck, Austria Evolutionary Biology,Zoological Institute, University of Basel,Switzerland ERIBA, University ERIBA, University CenterMedical Groningen, Netherlands The 1,2, †, Birgit Lengerer 1 , Eugene Berezikov , Eugene 3, ‡, Roberto Arbore Roberto ‡, 4 , Peter Ladurner , Peter gination and proofreading process, which which may process, and proofreading gination and undergone full and review buthas peer full undergone not

2,§ 3 , & Lukas Schärer Lukas , &

, Robert Pjeta , Robert 3 , Julia Wunderer , Julia 1 3 , Accepted Article ecological ecological conditions, and that these functionallyare torelevant key reproductive specific transcripts in indeed function in gametogenesis pathways. Weconclude that largea proportion of sex- these that establishes candidates ovary-specific and testis- selected of interference RNA thatconfirm manyplastic transcripts exhibitthe expected organ-specific expression,and whole-mount Using level. competition sperm increasing expectedthe shift away female from and towardsreproductive maleinvestment with transcripts (seminal fluid candidates). This transcriptional response closely corresponds to gonad-specific transcripts (spermatogenesis and oogenesis candidates) and tail-specific differentially expressed according theto social environment, rising to>30% ofputative we demonstrate that at least 10% of the >75,000 investigated transcripts in on RNA-Seq data from 16 biological replicates spanning four different group size treatments, simultaneousmodel hermaphrodite,free-livingthe flatworm a in plasticity allocation sex such for test to assay expression gene genome-wide a performed we Here success. reproductive for male competition of sperm importance increasing the to due size, group increasing with favoured is function male the into investment greater –that supports evidence morphological – and predicts Theory functions. sex female versus male the into investment relative the describes hermaphrodites simultaneous environments. Acrucial life history trait that is often highly plastic is sex allocation, which in Phenotypic plasticity can enable organisms to produce optimal phenotypes in multiple Abstract § This article by article rightsprotected This reserved. is All copyright. plasticity. allocation phenotypes. Our study thus begins to bridge organismal and molecular perspectives on sex Oeiras, Portugal Current address: Instituto Gulbenkian de Ciência, Ruada Quinta Grande 6, P-2780-156 M. lignano are differentially expressed according to the prevailing prevailing the to according expressed differentially are in situ

Macrostomum lignano hybridization, we then then we hybridization, M. lignano . Based . Based are Accepted Article (Janicke relevant environmentalcues of matinggroup size (Schärer 2009) –such as social group size favourthe evolution phenotypically of plasticsex allocation strategies respond canthat to competition in determining fitness gains through the sexmale function. This thoughtis to plus one)increases (Charnov1982), to the due increasingimportance of successin sperm towards the male functionsex as the mating group size (i.e. the number of mating partners more investment shift individualsthat should indicates Theory 1996). 1982; 1979; (Charnov the balanceresources of invested into individual's an maleversus its female sex function In simultaneous hermaphrodites, the question of optimising sex allocation means predicting simultaneous hermaphrodites(Munday and sequential in also adjustments sex allocation for support experimental accumulating studyingsex ratios separate-sexedin taxa (Hardy 2002; West 2009),therenow is Frank 2000; 2002). there Although is strong a inthesexbias allocation literature towards heralded as one of the great success stories in predicting adaptive evolution (e.g. West 1982; West 2009),the and widespread empirical support for sex allocationtheory is often Charnov 1967; Hamilton 1930; Fisher 1884; Düsing 1871; (Darwin biology evolutionary of concern central been a long has allocation sex organism’san optimal Predicting Introduction competition Sperm Spermatogenesis; allocation; Sex plasticity; Phenotypic Oogenesis; expression; Gene Keywords This article by article rightsprotected This reserved. is All copyright. Schleicherova Tan 2003; Ladurner & Schärer et al. et 2013) – for which there is strong empirical support (Trouvé et al. 2010; Al-Jahdali 2012; Janicke et al. 2004; Brauer Brauer 2004; et al. 2006; Schärer 2009). Schärer 2006; et al. et al. et 2007; Schärer & Janicke 2009; 2009; Janicke & Schärer 2007; 2013). Because in this manner they

et al. 1999; et al. et

Accepted Article adjust theirsex allocation prevailingto suit the conditions (Brauer flexible response, i.e. even adult worms moved to new a social environment can rapidly (Janicke increasing group size is in close agreement with predictions from sex allocation theory atlarger2013) group sizes. relative Such towards shifta sexwith thefunction male hermaphrodites,we performed anRNA-Seq experimentin understood (Pannebakker perspective,the genetic detailshow sex of allocation plasticity is achieved remainpoorly a from morphological theory allocation sex substantiating ofevidence body this Despite theory. allocation sex test to of doing so, this makes simultaneous hermaphrodites especially valuable and unique models payoffs fitness the receive and allocation sex their adjust both lifetime, own their within can, This article by article rightsprotected This reserved. is All copyright. (Schärer 2007). Responsesinsize ovary tendtobemore variable, is typicallyit but either reduced (Giannakara spermatogenic cells(Schärer Ladurner 2003; Brauer sperm production. andegg largergroupAt sizes,worms exhibit largertestes (Schärer& because it is known to respond to relevant social cues by plastically adjusting its allocation to plasticity, sex allocation of the genetic basis studying for model an excellent represents living, simultaneously hermaphroditicflatworm (Ladurner et al. et et al. et al. 2013). Moreover, this shift has been shown to represent phenotypicallya 2005) or sometimes unaffected (Schärer & Ladurner 2003; Janicke 2016), and so have a higher sperm production rate (Schärer & Vizoso et al. et et al. 2007; Janicke et al. 2011).begin toaddress simultaneousfor To case this of the 2004b) and exhibiting a faster rate of spermatogenesis et al. 2013) containing more active active more containing 2013)

Macrostomum lignano et al. 2005b). This species species This 2005b). et al. 2007). et al. , a free- , a

Accepted Article covering of algae, and allowed to lay eggs. Three days later, all parental worms were parental DV1 worms were distributed among 12 Petri dishes containing 20ml f/2 and a dense experimental subjects. To generate experimental subjects of standardized age, ca. 1100 adult We began by confirming sex allocation plasticity at the morphological level for our size Group experiment transcriptomeand assemblies for thisspecies (Wasik (Janicke line DV1 inbred highly 1988; Ladurner curvilineata f/2, nutrient-enriched a artificialseawater (32‰ salinity),fedand with diatoms ( with filled dishes Petri inglass maintained are cultures Worm as an adult. inlength 1.5mm M. lignano organismStudy Methods & Materials experiments. interference RNA and hybridization of combination a using characterizations functional with up this followed then transcriptional landscape of sex allocation plasticity. For selected candidate transcripts, we predictedto different lead to optimalsex allocation patterns, we here aimeduncoverto the oroctets), pairs isolated, sizes: (group environments social in different individuals raising By This article by article rightsprotected This reserved. is All copyright. of batch each from hatchlings resulting the harvested We removed. were parentals the later days three then and way same inthe prepared dishes Petri of batch second a to transferred is a transparent, outcrossing simultaneous hermaphrodite that reaches ca. ), on a14:10 light:dark cycle at 20°C and 60% relative humidity (Rieger et al. 2005b). Allanimals in this experiment were taken from a culture ofthe et al. et 2013) that has also been used to generate the genome et al.

2015; Wudarski et al. in situ 2017). 2017). Nitzschia et al.

Accepted Article hatching in to laying egg from time Development introduced. were adults the after days ten dishes This article by article rightsprotected This reserved. is All copyright. differential sex allocation: differentfour treatment groups,representing differentsocial contexts expectedto tolead On the first day of the experiment (day 0), 1016 hatchlings were allocated randomly to one of theat start of the experiment was 2-5 days. the differentthe treatmentgroups, waswhich nevertheless expected tobe suspension, the amount of which was adjusted to equalize per capita food availability across well plates (TPP, Trasadingen, Switzerland) containing 1.5ml f/2 and fed with densea algae duration of the experiment. experimental All groups were constituted in single wells of 24- individuals, in 38 groups), in which worms were kept together in groups of eight for the which two worms were kept together for the duration ofthe experiment; and studying short-term responses to mating); at (aimed together paired were worms two always 24h final the for that except experiment, experiment;the the the group size experiment into four independent batches, and then within each batch we independent biological replicates for downstream analysis, wesplit the 28 plates used during into worms the sort then To section). next (see allocation sex of assessment morphological maximally one worm per for well the day subset37-3864,On a of wormstreatment per groupwas randomly selected (taking treatmentsame group on samethe plate. in total).On day63, position within plates. Worms were transferred to fresh wells every 7-11 days (five transfers this required 28 x 24-well plates, with treatment groups balanced across plates and for M. lignano joined joined (n = individuals)224 were kept alone for the duration of the is around five days, meaning that the expected age of the hatchlings isolated worms were pairedrandomly with a chosenindividual fromthe (n = 224 individuals) were kept alone for the duration of of duration the for alone kept were individuals) (n= 224 pairs and pairs pairs octets (n = 264 individuals, in 132 groups), in to ensure statistical independence) for

ad libitum ad octets . In total, total, . In (n = 304 304 = (n Accepted Article Source Imaging The 41BF02; DFK (model camera Firewire digital a and microscope DM2500 Leica a using magnification 400x at ovaries) (testes, interest of organs for obtained then squeezed a microscopeon slide to a standardthickness using spacers, and photomicrographs and ovaries. Images were later processed with ImageJ (http://rsb.info.nih.gov/ij/) to obtain dimensional images thereby obtained correspond well to the tissue volume of both the testes ProBTV (Ben Software). Due to the standardized squeezing, we expect that the two- (Schärer & Ladurner 2003). Briefly, worms were relaxed using a solution of MgCl of solution a using relaxed were worms Briefly, 2003). Ladurner & (Schärer species this for techniques standard using measured were worms of ovaries and testes the function, sex female the vs. male the into investment relative the estimate to order In Morphological sex allocationassay of plasticity RNA -80° extraction. until stored at homogenized using bead a beater in 700µl of cold (4°C)TRIzol® Reagent (Invitrogen)and shown (Janicke much more male-biased sex allocation than the next closest group size of two, as has been a predict still would seven of size a group that reasoned We used. be still could wells these in worms seven remaining the that so groups), 6/38 (in tolerated was well per worm single lost. However, to avoid losing large numbers of groups, in the case of the where anindividual diedthe in worms contained,of it from 49-76 individuals (median: individuals). 57 that Notecasethein our wormcultures (here around 6%), eachbiological replicate differed slightlynumberthein in observed normally mortality background of level and a low contents inplate differences independent biological replicatestheforexpression gene measurements. Owing toslight treatmentand batch were pooledinto just replicate).one total,In we thereby produced4 4x pooled all of the worms belonging to the same treatment group (i.e. all worms from the same This article by article rightsprotected This reserved. is All copyright.

Europe GmbH, Bremen, Germany) in combination with the image capture software et al. 2013). Whole worms for each replicate sample were combined and and combined were sample replicate each for worms Whole 2013). isolated , joined or or pairs treatments, the whole replicate was was replicate whole the treatments,

octet s

the loss of a a of loss the 2 and then Accepted Article in and ovary area are highly repeatable and have been employed in numerous previous studies allocation testis as area (testis/ +ovary area area)(Suppl. S1). File These measures oftestis estimatestestis of area and ovary area for each worm,from which weestimated sex This article by article rightsprotected This reserved. is All copyright. all all libraries are based on extractions from whole worms, such that the resulting read counts SA (Plan-les-Ouates, Switzerland) using their library construction. Library construction and subsequent NGS was performed by Fasteris for allother samples,amount the ofRNA starting material was equalized to 500ngprior to Qubit (picogreen) were in the range 590-1400 ng, except for one sample assessed at 330 ng; (i.e. astandard TRIzol extraction protocol for liquid samples). Quantitation assays using the protocol and finally re-suspended in 20µl of nuclease free water after removing the DNase water, free treated with RQ1RNase-Free DNase (Promega)following the manufacturer's nuclease of in100µl re-suspended protocol, extraction chloroform standard a using worms whole from extracted was RNA Total group). treatment per four (i.e., analysis transcriptomic treatment,per weindependentgenerated total 16 of a cDNA libraries forfurther pools RNA replicate four the on Based response. this of basis transcriptional the investigated adjust theirsex allocation prevailing to the social conditions’ Results the in nextsection), we they shift investment towards the male sex function with increasing group size, see ‘Worms environment by altering their sex allocation in agreement with our expectations (i.e. that Having shown that our experimental worms responded to their prevailing social Total RNA extraction and library construction Janicke Schärer 2003; Ladurner & Schärer question), butnot directly – in the case of tissue-specific transcripts – about whether such ustell about overall differences in expression between treatments (our primary research M. lignano et al. et 2013; Giannakara to compare sex allocation under different experimental conditions (e.g. et al. et al. 2016)). 2004b; Brauer 2004b; standard (Illumina) mRNA protocol. Note that that Note protocol. mRNA (Illumina) standard et al.

2007; Schärer & Vizoso 2007; 2007; & Vizoso Schärer 2007; Accepted Article mapping ambiguity. from the read mapping data by RSEM v1.1.19 (Li & Dewey 2011), which accounts for read total the of transcriptome(Suppl. Table S1).Next, transcript expression levelswerederived we we obtained between ca. 7.3 to10.1 x10 total of of 76,437 transcripts74,211 a to 97%) (= inMLRNA110815 the assembly;per sample, -best –strata”. Mapping yielded 132,607,805 mappablereads, with at leastread one mapping bowtie v. 0.12.7 (Langmead interest in this study. Sequencing reads were mapped theto MLRNA110815 assembly using female-specifictranscriptsand of male- therefore all contain expected to assemblies are juveniles at different stages of development and adult hermaphrodites of different ages. The transcriptome assemblieswere made from mixed populationsofanimals containing the for used libraries the sampling, transcriptome of diversity maximal the ensure assembled and annotated in a similar way, but was based on a smaller RNA-seq dataset. To novo published work (Arbore (http://www.macgenome.org/download/MLRNA110815/) for consistency with previously the to mapped were reads Sequencing (Fasteris). reads 100bp single-end 2000,sequencing HiSeq a of lane one from Following cDNA library construction andmult generationNext sequencing andmapping Results). in the section gametogenesis’ for norms reaction ‘Genomic inthe this question explore we (although volume unit per expression and/or volume tissue in shifts from stem changes This article by article rightsprotected This reserved. is All copyright. transcriptome assembly MLRNA150904 (Grudniewska (Grudniewska MLRNA150904 assembly transcriptome et al. M. lignano et al. 2015). MLRNA110815 is an early version of the reference reference the of version early an is MLRNA110815 2015). 2009) 2009) parameters with 2 “-n -e 9999999916 -l -a -m 200 -

de novo de 6 mappable reads, with coverage between ca. 79-85% transcriptome assembly MLRNA110815 MLRNA110815 assembly transcriptome iplexing, NGS sequence data were obtained

et al. 2016), and it was de de Accepted Article transcripts. tissue-specific of putatively expression differential investigate to able also were we 2015), (Arbore study RNA-Seq’ ‘positional published previously a from expression transcript combining our‘social RNA-Seq’ expressionwithdata datathe anatomicalon location of expression patterns, incorporating information from all four treatment groups. Moreover, by characterization, and the latter usrangeenablingtestto of specific fora differential number of transcripts as putativedifferentially expressed candidates for follow-up functional contrasting treatments groups,i.e. most two the between comparisons pairwise permitting theformer with complementary, Huber 2010)and We identified differentially expressed candidates using the programs Differential expression analysis value cutoff below 0.01, plus Pfam domains from the Pfam database v. 27 (Finn al. melanogaster (Wudarski extractedfrom the genome-guided transcriptomeassembly Mlig_RNA_3_7_DV1_v3 theand single best hits from both searches were merged. Functional annotations were then vs. MLRNA110815) Mlig_RNA_3_7 and vs. Mlig_RNA_3_7 MLRNA110815 (i.e. directions al. most recentgenome-guided transcriptome assembly Mlig_RNA_3_7_DV1_v3(Wudarski For assigning transcripts from the the from transcripts assigning For This article by article rightsprotected This reserved. is All copyright. 2016) 2017), sequences were mapped by blast with the parameters “-W 30 –e 0.01” in both inboth 0.01” –e 30 “-W parameters the with by blast were mapped sequences 2017),

identified using blastx v.2.2.6 (Altschul (Altschul v.2.2.6 using blastx identified et al. et (BDGP), (BDGP), 2017); these include homologs from human (GRCh37), baySeq Caenorhabditis elegans (Hardcastle & Kelly 2010). The two approaches are de novo de isolated transcriptome assembly MLRNA110815 tothe vs. octets (Wormbase) and and (Wormbase) et al. et , thereby identifying the maximum maximum the identifying thereby , 1997) and taking the best hits with e- with hits best the taking and 1997)

S. mediterranea DESeq Drosophila (Anders & & (Anders et al. (Brandl (Brandl 2016). et al. et et

Accepted Article samples), contrasting expression in the four replicates of the of replicates inthe four expression contrasting samples), was compared for ca. 72,000 genes (i.e. allgenes with count > 0for at least one of the eight anddesign estimatingdispersion parameterswhole basedthe on dataset,gene expression all count data was rounded to nearestthe integer value. After specifying the experimental (Anders & Huber 2010),implemented in Bioconductor v2.10 in Rv2.15.1. Prior to analysis, Differential expression (DE) analyses were first performed using This article by article rightsprotected This reserved. is All copyright. additional analysesinSuppl. Table S2.The qualitative conclusions remain unchanged. these also report completeness for but above, described as approach inclusive most the results main the in report therefore We treatments. between expression in consistently filtering),no filter outlow but might abundance transcriptsnevertheless that differ remain that total,9,070(in areDE called with as pre-filtering, 40% compared to7,584with thereby increases the yield of) differentially expressed candidates among those transcripts (and detect to power us more ofgiving advantage the has approach latter the replicates; expressedcandidates, especially given that we testfor differences based ononly 8 biological differentially identify comprehensively to attempt inour counterproductive be could which negatives, false of generating risk the runs but conservative, is more approach former The or 40%; see (Anders & Huber 2010)) of transcripts with the lowest overall expression level. stringent (=0.05)FDRcriterion and (ii) pre-filtering remove to fixed a percentage (either 20 exploredWe also the effectof twoadditional stepstheon analysis: (i) adoptinga more 0.1. of (FDR) rate discovery false a adopting testing, multiple for control to correction to assess each transcript (AndersHuber & 2010),followed a Benjamini-Hochberg by the four replicates of the

octets treatment. The test involves use of a negative binomial model

isolated DESeq treatment with that in version 1.8.3 1.8.3 version Accepted Article transcripts, i.e. DE i.e. transcripts, isolated) pairs, joined, =(octets, NDE DE DE DE DE DE DE questions:(1) whatproportion of transcripts differentially are expressed between research specific three address to classes DE relevant for transcripts of proportion estimated with posterior probabilities for each transcript for each class. We then summed the relevant program returnsestimates the proportion of transcripts of belongingclass, toeach together where treatments contained within samethe brackets do not differ in expression level. The DE patterns (DE expression.defined We eight classes: seven biologicallyplausible differentialexpression datasetbelongs certain toa ‘class’, where each definesclassa particular pattern of empirical Bayesian framework to calculate posterior probabilities that each transcript in the single analysis,to thereby test for specific differential expressionscenarios. into a incorporated be could replicates 16 biological all from information that so 2010), program the using parameters expression differential estimate Since analysesusing This article by article rightsprotected This reserved. is All copyright. the largest two and matinggrouptreatments size of treatment groups (NDE): G F E D C B A = (octets, pairs, joined), (isolated) (isolated) pairs, joined), =(octets, isolated) (pairs,joined, =(octets), isolated) (pairs),(joined, =(octets), (isolated) (pairs),(joined), =(octets), isolated) pairs), (joined, =(octets, = (octets, pairs), (joined), (isolated) (isolated) pairs), (joined), =(octets, (isolated) (pairs,joined), =(octets), A – DE A G +DE ) plus one in which there was no differential expression across all four DESeq E +DE can only beperformed a pairwise on basis, we soughtnext to G ); (2) what proportion of transcripts are differentially pairs

and octets baySeq (‘not mating vs. mating’ mating’ vs. (‘not mating (Hardcastle & Kelly BaySeq isolated uses an

Accepted Article DE an existing dataset reporting positional patterns of gene expression in expression of gene patterns positional reporting dataset existing an matched the differentially expressed candidates generated by the pairwise Finally, to generate tissue-specific candidates for functional characterization, wecross- asclear evidence for that specific patternof expression. this interpret would of0.95, we probability a posterior exceeds itself DE by class particular evidenceforthe differential expression specificof transcripts.Moreover, where one patterns, the sum of posterior probabilities for different DE classes can also be used judgeto Although the and identified more than 4,000 transcripts changing in expression between adjacent samples – samples adjacent between in expression changing 4,000 transcripts than more identified or tail regions, respectively, and making these transcripts good candidates for having the the having for candidates good transcripts these making and respectively, regions, tail or i.e. Bvs. A, C vs. B, or D vs. C – indicating an enrichment for expression in the testis, ovary, worms).Using differential a expressiona log criterion of head, testis and ovary regions, and (D) the head, testis, ovary and tail regions (i.e., whole RNA-Seq containing either (A) only the head region, (B) the head and testis regions, (C) the for pools RNA four generated they regions, these between boundaries the to corresponding fragments into worms cutting By overview). a morphological Fig. for 4A see organs; seminal fluid-producing prostatecells gland – tail and the plateits with associatedadhesive region(containingtail developing genitalia femaleincluding– male and eggs, the the thewhichspace on both gutisof primarily sidesthe occupied the paired by ovaries) andthe space on both sides of the gut is primarily occupied by the paired testes), the ovary region (in rostrum, eyes, brain and pharynx with associated glands), the testis region (in which the expression in four different body regions of the worm: the head region (containing the al. expressed between This article by article rightsprotected This reserved. is All copyright. 2015). Briefly, Arbore et al. generated tissue-specific candidates by examining gene F ); and (3) what proportiontranscripts of are differentially expressed between joined worms (‘early response’ to mating transcripts, i.e. i.e. DE transcripts, to mating response’ (‘early worms baySeq pairs pairs analysis was primarily implemented to estimate these broad scale and octets (‘mating groupsize’ transcripts, i.e.DE 2

-expression change >2, this B + DE + M. lignano E +DE DESeq B + DE F + DE analysis to (Arbore (Arbore isolated C + DE + G )? )? D

+ et Accepted Article Whole-mount In situhybridization screen accountfor >99% of all transcripts. additionalof classes werealsodefined Arbore by Anumber expressed. ubiquitously are transcripts these that implying necessarily than rather that this simply thatmeans we do not yet have any evidence for tissue-specific expression, i.e. class [0,0,0]. Following Arbore regions, body different the between differ substantially not did that those were transcripts also interpret both classes as likely ovary-specific candidates. By far the largest class of social group sizes of classes [0,+,0] and [+,+,0] inour study (see below), and we therefore classificationfurther isnow supported the by highly similar expression responsesdifferentto (Arbore fragments (B) testis the in material ovary of contamination some likely was there that fact the to owing candidates ovary-specific as interpreted also were [+,+,0], class i.e. comparisons, sample B C vs. and A vs. B the both in expression candidates as [0,0,+]. Notethat members of a further class of transcripts that increased in designatedwere class as [+,0,0], ovary-specific candidates as [0,+,0]tail-specificand pattern for these three fragment comparisons, meaning putative testis-specific candidates somecases). All transcripts were assigned to positional classes according tothe expression by validated was (which expression tissue-specific corresponding This article by article rightsprotected This reserved. is All copyright. We attempted toinvestigate patterns of expression for a set of 15 transcripts previously the primers specified in Suppl. File S2) according tostandard protocols in are are and not are differentially expressed in different social environments, and toinclude a identified testis-specificas candidates, haphazardly sampled to include both transcripts that (Lengerer et al. in situ 2014). hybridization (ISH)performed was fortotal a 97 transcripts of (using et al ., wehere term these ‘non-tissue specific’, but note et al ., but collectively the above five classes classes five above the collectively ., but

in situ et al. hybridization in in hybridization 2015). This M. lignano

Accepted Article candidates (MLRNA110815_14701, MLRNA110815_21744, MLRNA110815_24606, MLRNA110815_24606, MLRNA110815_21744, (MLRNA110815_14701, candidates RNAi was performed for 12 putative testis- or ovary-specific, differentially expressed screen RNAi imagesand Mowiol were taken using a DM5000 Leica microscope. in mounted were °C. Specimens 37 at (Roche) system NBT/BCIP the using developed was were used. Anti-digoxigenin-AP Fab fragments (Roche) were diluted 1:2000 andthe signal digoxigenin-labeled RNA probes, polymerase T7 (Promega) and DIG labelling mix (Roche) casesan additional time with different a batch of cDNA.synthesize To single stranded most in and temperature, annealing different a using once least at repeated was it failed, DNA was produced using standardPCR reactions. Infor caseparticular a transcriptPCRthe Template primers. reverse the end of 5' the at added was region promoter a T7 and 0.4.0/) In each case, primers were designed with Primer3 software (http://bioinfo.ut.ee/primer3- represented. over- to be appears types tissue or organs particular in expression whether ascertain begin to probe the potential sites of expression of this large class of transcripts and to well as 63transcripts from the non-specific class. These latter transcripts were included to as classes, ovary-specific the from 19 transcripts sampled similarly We specificity). probe ISH),with thatwell as as transcriptthe length >300 hadbe to base pairsfacilitate(to ISH assess to difficult are transcripts expressed weakly (because datasets RNA-Seq study) (this represented by at least ca. 100 reads in both the ‘positional’ (Arbore overallrange of expressionlevels, withthe qualifying criteria that transcript the had tobe This article by article rightsprotected This reserved. is All copyright. MLRNA110815_35903, MLRNA110815_10574, MLRNA110815_1085, MLRNA110815_54, MLRNA110815_13829.2, MLRNA110815_3206.2, MLRNA110815_1214.1,

et al. 2015) and ‘social’ ‘social’ and 2015) Accepted Article the wholethe experiment, animals werefed transferred clean to a embryo glassdish and thedsRNA solutionwas changed.Throughout were animals hours, 24 Every dish). per solution of 400µl (in dishes glass embryo hatching and were maintained for three weeks. For each transcript, 30 animals were kept in prevent the selection of resistant bacterial strains. Treatments started from one week post- to day third every alternated were µg/ml) (50 Ampicillin and Kanamycin, Streptamycin, concentrationng/µl. 15 solution of supplemented The was withwith algae and antibiotics. scale RNA kit, Promega). DsRNA was diluted in artificial sea water (ASW) to afinal system using primer pairs with SP6 and T7 promoter regions (T7 and SP6 Ribomax™ large Briefly, double-stranded (dsRNA)RNA probe was generated by an experimental subjects (prepared in a similar way as in the initial group size experiment). al. why these particular transcripts were selected), plus a positive control (macif1 (Lengerer MLRNA110815_10265 and MLRNA110815_26815.1 – see Results for further explanation This article by article rightsprotected This reserved. is All copyright. assessed using ANOVA, adopting Dunnett’s method for comparing multiple means to a was control, negative the to relative transcript, each for effects treatment of significance make this difficult to discern). Forbody area, testes area, ovary area and egg number, the recordedthirdand ‘unknown’ a since as of level – in presence the the antrumcan eggs of magnification for the presence or absence of sperm, which was sometimes inconclusive – 400x at the antrum examining by (assessed status sperm received and imaging) plus body area (at 40x magnification), egg number (counted under the microscope during underthe microscope toassesstestes area and ovary(measured area described as above), for phenotypes assessed morphologically then were worms The ISH. performing by verified culture conditions. Atthe end of the experiment, the efficacy of the RNAi knockdown was Fisher Exact Tests (Freeman-Halton extension) with a Bonferroni correction to to the correction Tests(Freeman-Halton Exact extension) with a Fisher Bonferroni 3x2 of means by assessed was significance status, sperm received For mean. control single 2014)) and a negative control (no dsRNA), using standard protocols and anew batch of ad libitum and were maintained under normal normal under maintained were and

in vitro transcription et Accepted Article isolated allocation (Brauer et al. experiment. In the of end the to h prior 24 for treatment same the from worm such another with paired then termed way ANOVA, (one- allocation sex for proxy our on effect significant a highly size had group predicted, As Worms adjust their sex allocation to the prevailing social conditions Results confirmationpending withfully replicated a experiment. treatment phase. The specific results obtained should thus be interpreted with due caution, wormsall receivingtreatment same the were inhousedembryo same the duringthedish screen, prospective initial, this for that note We control. negative single the to comparisons ( threshold significance This article by article rightsprotected This reserved. is All copyright. Having shown that our experimental worms responded to their prevailing social The socially of sensitive lignano transcriptome M. (McGraw mating of commencement upon expression in change that genes response’ ‘early putative allocation,sex with worms in raised in raised worms and allocation sex female-biased most the investigatedtranscriptional the basisthis of response. Beginningwith the pairwise environment by altering their sex allocation in agreement with our expectations, we next 2004a), butnotlong enough for wormsto adjustsignificantly their morphologicalsex joined and et al. joined F , involved, worms beinginitially raised pertheas 2004; 2008) (see‘Transcripts affectedby matingstatus’ below). 3,145 M. lignano = 61.75, =61.75, et al. worms in Fig. 1). The The 1). Fig. in worms α 2007) (as evident 2007) is also greatly from the overlappingSEsbetween = 0.05/12 = 0.004) account to for the treatmentmultiple , this should belong enough toensure multiplematings (Schärer P < 0.0001, Fig. 1): worms raised in the inthe raised worms 1): Fig. 0.0001, < pairs joined being intermediate. The fourth treatment group, group, treatment fourth The intermediate. being treatment group was included to identify

octets isolated had the most male-biased male-biased most the had isolated treatment group, but but group, treatment treatment had Accepted Article next performednext below ‘Genomic (see reaction norms for gametogenesis’),butlimitation toremedy the we allocation continuum. This assumption is actually justified by subsequent analyses presented treatments,implicitlywe that assumed thesetwo groups represent two extremessex ofa By initially focusing on just the the mainof organisms studied todate. unannotated, likely reflecting the phylogenetically distant position of important pattern that emergesfrom this analysis is that many plastic genesremain plastic candidates, we did not restrict ourselves to only annotated genes, because one noteAlso that in illustrating (where possible) the functional annotations of the top 100 S5. File in Suppl. provided 2 are Fig. produce to used annotations complete the and S4, and S3 Files in provided Suppl. are assemblies these between inter-converting for – details here annotations comefrom a more recent transcriptome assembly than the one we employed lignano in Fig.2, alongside functional annotations extracted from the most recent genome-guided (based on statistical support for differential expression, i.e. adjusted p-values) are depicted candidates plastic 100 top the for patterns expression plasticity, this of some illustrate To (Suppl. File S3). transcripts3,082 exhibit significantly or lowerhigherexpression in putatively differ in expression according social tothe environment: specifically, 4,503and that transcripts candidate 7,500 over generating expressed, differentially are transcripts the the expression data, which is explored in the next section—our global estimate from comparing comparison of the two most extreme treatment groups—and for now ignoring positional This article by article rightsprotected This reserved. is All copyright. octet transcriptome assembly (Wudarski (Wudarski assembly transcriptome and isolated baySeq treatments using using treatments analyses that allowed us to incorporate more complex differential differential complex more incorporate to us allowed that analyses octet and DESeq isolated isolated et al. is that 9.9% ofthe 76,437 investigated 2017). Note that this means the the means this that Note 2017). treatments, ignoringand the intermediate

octets M. lignano , respectively , respectively from some from M. Accepted Article could be identifiedcould be by identified differentiallyas expressed, with 92.4% of differentially expressed candidates that DESeq however, that there was a strong overlap between the rather to estimate the proportion of transcripts belonging to each DE class. Wenote, The transcripts expressed differentially specific identify to aim not primarily did analysis this cases, In all below.) joined group size’ transcripts, Fig. 3C)? (Note that a third comparison – contrasting between expressed differentially research questions (Fig. 3B-D). First, we asked what proportion of transcripts are summed3A), theseestimatedwe proportions relevant for classes DE to address specific the whole transcriptome belonging to the range of different DE classes described above (Fig. about the nature of expression differences. Having estimated the proportion oftranscripts in expression patterns from allfour treatment groups, andthereby test specific hypotheses This article by article rightsprotected This reserved. is All copyright. octets with an additional 8.39% (ca. 6250 transcripts) differing in expression between which transcript expression in estimated that 10.63% of transcriptomethe (ca. 7950 transcripts) belong to aDE class in around one fifth of all transcripts – is socially sensitive in its expression. Specifically, we proportion of transcripts are differentially expressed between pairs

baySeq and treatment groupscovered 'Transcripts the in –is affectedby mating status'section (Fig. 3C). comparison described above with respect to the transcripts that these analyses octets analysis confirms that a substantial portion of the (i.e. ‘not mating vs. mating’ transcripts, Fig. 3B), and second, what per se per baySeq (astatistically more difficult task,Hardcastle Kelly & 2010), but also identified as such by identified suchas also isolated isolated worms differs from that in from worms differs and the two largest mating group size treatments of of treatments size group mating largest two the and baySeq DESeq DESeq

analysis andpairwise the pairs pairs M. lignano (data not shown). not (data pairs and octets and transcriptome– octets isolated (i.e. ‘mating ‘mating (i.e. pairs pairs (Fig.3B), and and Accepted Article remaining 17 positional classes defined in Arbore expression pattern. For completeness, a summary of differential expression according to the that – based on the data farso available – we have so far found no evidence for a specific belongs to class [0,0,0], does this not necessarily mean that it is ubiquitously expressed, only sex allocation response to group size. We also emphasize again that just because atranscript the putative that given tissuespecificity refersto those tissues likely most involvedbe toin a threeleast likely times as socially-sensitivebe to their in expression, might as expectedbe expression in particular body regions (see below). Thus, tissue-specific candidates appear at higher exhibit to shown been previously had that classes tissue-specific putatively the for expressed differentially were transcripts of >30% typically contrast, By transcripts. specific class,so this in this and socially-sensitive fraction represents still only non-all <10% of expression in larger groups (Fig. 4B). However, there are a very large number of transcripts (n= 2826) lower and 3302) = (n higher having transcripts of numbers equal approximately [0,0,0] did that not greatlydifferexprein the largest number of DE transcripts identified here belong to the non-tissue specific class far by Methods), and Materials (see study earlier an in identified classifications positional worm's anatomy (Fig. 4A). When splitting our pairwise expression, basedthe on positional separationof the male and femalesex functionsinthe expressed in particular body regions and thus having putatively tissue- and sex-specific predominantly transcripts for responses gene expression investigate to sought next We Defining transcriptional tissue-specificsize to altered group responses This article by article rightsprotected This reserved. is All copyright. significant increase expression in the respective of transcript bodyregion the in containing the from solely stems tissue-specific putatively as assignment the case, each in that ovary-specific (classes [0,+,0]and [+,+,0]) tail-specific or (class [0,0,+]).Recall, however, transcripts that represent strong candidates for being either testis-specific (class [+,0,0]), in the next sections, we focus solely on these three main putatively tissue-specific classes, i.e. potentially involved in gametogenesis, we have not yet confirmed whether the transcripts are maleorfemaletissuethe interest, of the for majority and so transcriptsof identified being as ssion between different body regions, with et al. (2015) is given in Suppl. Table S3, but S3, but Table Suppl. in is given (2015) DESeq DESeq

dataset according to the Accepted Article expressionchanges purelywere with this line in morphologicalresponse, we would expect all transcript the if So, octets. in volume testis in increase 1.56x a implying respectively, observed substantial heterogeneity in the expression changes (with fold-changes ranging ranging fold-changes (with changes expression the in heterogeneity substantial observed transcripts to change in expression by ca. 1.56x. This is clearly not the case, since we isolated quite closely reflects the changes in testis volume occurring between treatments. Comparing truly testis- or ovary-specific (but see ‘ This article by article rightsprotected This reserved. is All copyright. about in changes in gonad size. Taking the testis as an example – arguably the organ we know most thatunlikely expressionthe changes wehave documentedcan beaccountedfor purely by seems it Nevertheless, tissue. ovarian and testicular of amount in the shift relative the by for spermatogenesislargerin groups, i.e.their expression pattern is atleast partially accounted towards and oogenesis from away switch the represent directly extent some to presumably 4E). These expression responses by putatively testis- and ovary-specific candidates majority of these (200/205 = 98%) exhibit lower expression in larger groups (Fig. 4D and [+,+,0] exhibited evidence for differential expression (i.e., ca. 46% overall), but the vast class the to belonging transcripts candidate ovary-specific of 63%) i.e. ca. 127, of (out 80 and (out of 323, i.e. ca. 39%) of ovary-specific candidate transcripts belonging to the class [0,+,0] majority of these (1033/1053 = 98%) had higher expression in larger groups (Fig. 4C). 125 belonging to the class [+,0,0] exhibited evidence for differential expression, andthe vast molecular perspective: 1053 (out of 3360, i.e. ca.31%) of testis-specific candidate transcripts a from visible clearly also is plasticity allocation sex response, allocation sex morphological above-reported the on based expected and theory allocation sex by predicted As gametogenesis Transcriptomic reaction for norms below). candidates’ expressed differentially and M. lignano octet worms, found we on average testis areas of 9616 mm – the two-dimensional area we measure in squeezed worms probably In situ hybridization screen for tissue-specificity of tissue-specificity for screen hybridization

2 and 14981 mm 2 , Accepted Article isolated isolated thewith morphological response, i.e. the greatest difference in expression levels is between demonstratea quantitative shift in gene expressionisthat in close qualitative agreement clearly which section), next in the discussed are candidates tail-specific 5A-C; (Fig. candidates ovary-specific and testis- expressed differentially all for norms reaction specific tissue- as visualized be also can allocation female and male for responses contrasting The oogenesis. and spermatogenesis of regulation particularly interesting transcripts for functional characterization in the context of (negative) represent might octets) in expression higher had that candidates ovary-specific putatively the morphological response. These ‘exceptional’ transcripts (plus the corresponding five that had significantly lower expression in octets, i.e. the opposite trend to that predicted by morphology alone (meanfold-change 2.14x, median 2.02x).We alsoidentified transcripts20 excess of 1.56x), with an average change somewhat higher than expected based on 0.02 tofrom 7.08, 94% and differentially of ex This article by article rightsprotected This reserved. is All copyright. tissue-specific transcriptional responses we observe are in line with the graded graded the with inline are observe we responses transcriptional tissue-specific e.g. (Causton stressors, obvious more with systems other in observed been sometimes has (as expression gene of downregulation widespread in resulting response stress a generalized induce might isolation social of treatment the that namely data, our of interpretation possible one against strongly socially sensitivegenes, this gradedresponse across successivetreatment groups argues Together with the disproportionately large number ofsex-specific transcripts in the pool of 5B),there as isalso for the 80putative ovary-specific candidates in class[+,+,0] (Fig. 5C). there is a clear and complementary negative trend ofapproximately equal magnitude (Fig. with an approximately two-fold (i.e. one log putative testis-specific candidates the prevailing trend is clearly positive with group size, to and octets octet (Fig. 5A). For the 125 putative ovary-specific candidates in class [0,+,0] [0,+,0] in class candidates ovary-specific putative 125 the For 5A). (Fig. worms, with intermediate expression levels in levels expression intermediate with worms, et al. 2001; Levine 2001; et al. 2 unit) average increase in expression from from expression in increase average unit) pressed transcripts exhibiting fold-changes in 2011; Yampolsky 2011; Yampolsky

pairs et al. . For 1,053 . For the 2014)). Instead, the Accepted Article remaining transcripts, conducted as part project of a seminalon fluiddiversity andfunction (Arbore MLRNA110815_9549.4 and specific expression was alreadyreported assignment hasin many cases now been confirmedby wellas as by the striking degree of plasticity in their expression. Importantly, this functional those involvedinthe other major aspect of male allocation (i.e.testis-specific genes; Fig.5A), conclusionis based onthe similar reactionnorms for mostoftranscripts these compared to seminal fluid production, previously a unquantified component of male allocation.This in involved genes prostate-specific for candidates strong thus are size, group with expression Weber 2010; fluid substances added to sperm in the ejaculate (Ladurner exhibitmight such anexpression patternthe are prostate cells, gland which produceseminal candidates). The obviousmost morphological features located in the tail of wormsthe that ovary-specific and testis- for difference two-fold above-mentioned the (cf. groups social contrasting most two the between expression in difference seven-fold approximate of marked differential expression according to the social context (Fig. 4F, Fig. 5D), with an of the transcripts with putative tail-limited expression (150/366 = ca. 41%) exhibits evidence likely also reveals novel aspects of sex allocation in gonadsresult asa shiftofa in investment betweenspermatogenesis and oogenesis,our study In addition to characterizing the quantitative changes in gene expression that occur in the allocation of male aspects Novel Janicke 2009; Janicke & (Schärer experiments and previous inthis found response allocation sex morphological This article by article rightsprotected This reserved. is All copyright. in 76 whichof 76 expressed are exclusively in prostate glandcells(Weber etal.2018). M. lignano et al. , has revealed that ca. two-thirds indeed exhibit prostate-specific expression, 2018). These 150 transcripts, and especially the 140 that increase in et al. 2013). et al. for MLRNA110815_22046, MLRNA110815_80.4 2015), and a comprehensive screen of nearly all all nearly of screen comprehensive a and 2015), M. lignano in situ

et al. hybridization data: prostate- data: hybridization . Notably, a large proportion proportion a large . Notably, 2005b; a; Vizoso a; Vizoso 2005b; et al. et

Accepted Article individual transcriptsin the relatively little power toidentify them with our experimental design. Infact, only three number transcriptsof thatdifferentially are expressed accordingto matingstatus, we had DE classa (Fig. 3D). However, although this estimate suggests that there are a considerable baySeq of mating,onset we contrasted Finally, to examine putative ‘early response’ genes whose expression changes following the Transcripts affected by mating status This article by article rightsprotected This reserved. is All copyright. gene NCAPH, non-SMC Icomplex, condensin NCAPH, gene non-SMC H) subunit (Uhlén MLRNA110815_34533.1exhibits (which significant testis-enrichedhomologyto the human MLRNA110815_2487.2 exhibits(which significant NRDE2)homology andgene tohuman 0.95 allowing them to be unambiguously assigned differentiallyas expressed candidates. expression in MLRNA110815_892.8 (no significant human homology) exhibited a ca. two-fold lower whereas conclusion) this in caution urges former the of level expression overall highly expressedin non-specific classes. classes. non-specific subset of transcripts sampled from across the distributions of putatively testis-, ovary- and performedwe an genes, gametogenesis in plasticity tissue-specific likely of interpretation our validate to and To begin to probe the potential functions of the identified differentially expressed transcripts candidates differentially tissue-specificityfor of expressed screen In situhybridization analysis suggests 0.37% of all transcripts (i.e. around 280 transcripts) belong to such isolated in situ isolated worms compared to the other treatments. treatments. other the to compared worms hybridization (ISH) characterization of the sites of expression for a a for ofexpression sites of the characterization (ISH) hybridization wormscompared the to other treatments (thoughvery the low baySeq baySeq isolated analysis passed the posterior probability threshold of of threshold probability posterior the passed analysis worms to the remaining threetreatmentgroups.remaining The the worms to

et al. 2015) were both more more both were 2015) Accepted Article transcripts expressed in the testis region, Arbore et al. (2015) determined that four out of six six of out four that determined (2015) al. et Arbore region, testis in the expressed transcripts (Arbore Our differential expressioncalls canbecompared also topreviously reportedISH patterns Y). expressedinthe ovary and indeveloping eggs periphery). For the three candidates that are not socially sensitive in their expression, oneis testis the at or testis the in staining weak exhibiting further but ovary, the in expressed specific indeed exhibiting ovary-limited expression (and the three others also being patterns support this contention, with seven out of the ten transcripts expected to be ovary- whereas the remaining three would be less likely to be ovary-specific. Again, ISH the truly ovary-specific (because of their differential expression in candidates.cases,13 leaving could We predictthat these would ten of be likely more tobe candidates (n=7 from class [0,+,0] and n=12 from class [+,+,0]), for which PCR failed in six Similarly, for the ovary we attempted to investigate 19 transcripts identified as ovary-specific predictions, with all but one fitting the expected pattern (Fig. 6A-K). presumably less likely to be testis-specific in their expression. ISHThe data support these remaining candidates that did not show socially-sensitive differential expression are expressed candidates would show testis-specific expression. Bycontrast, the two other differentially eight the that predict could we transcripts ten remaining the for PCRs, failed transcripts could not be investigated due to the inability to design suitable primers or due to identifiedtranscripts previouslytestis-specificcandidatesas (Arbore withBeginning the testis, attempted we toinvestigate patterns of expression for aset of 15 This article by article rightsprotected This reserved. is All copyright. patterns). Of these four transcripts, three show strong testis-specific staining and are (as ISH on (based testis-limited actually were data) RNA-Seq positional on (based candidates et al. 2015), and these toolargely support our conclusions.main firstthe Taking and expressed twoare in gonads both (Fig. 6L-

isolated et al. and 2015). Whilefive octet worms) worms) Accepted Article S1). Fig. (Suppl. organs reproductive in expression specific exhibited revealing wide rangea ofexpressionpatterns, within including manyeven that class this of candidates, we selected a subset of 63 transcripts for exploratory characterization by ISH, classto [0,0,0]. begin probe To to potential the expression patternsthis of very largenumber expression in the earlier positional RNA-Seq study (Arbore socially-sensitivegene expression were those thatclear show not a did pattern of differential Finally, as described above, by far the biggest class oftranscripts found to exhibit evidence of (MLRNA110815_12337.1). differential expression in the same direction (MLRNA110815_6266) and the other does not For the two remaining transcripts for which no ISH pattern was obtained, one exhibits dataset. inour expression differential predicted the of evidence show (MLRNA110815_7498) MLRNA110815_7498, MLRNA110815_2640 and MLRNA110815_4558) andall but one MLRNA110815_7725.2, MLRNA110815_1618.1, (MLRNA110815_16738, candidates investigated indeed had ovary/developing egg-limited expression (MLRNA110815_9262) is not. For the ovary region, six out of eight putatively ovary-specific other the and analysis our in expressed differentially is (MLRNA110815_10311.2) limited in their expression pattern (but rather expressed in the gut epithelium), one testis- be to not (2015) al. et Arbore by found candidates region testis two remaining the Of study. in our differentialexpression sensitive socially of evidence no shows and experiments transcript, MLRNA110815_3228, was found to exhibit a weakly testis-specific signal in ISH fourth A MLRNA110815_6628.2). MLRNA110815_9973.1, (MLRNA110815_7008, data RNA-Seq social on our based sensitive socially also predicted) be might This article by article rightsprotected This reserved. is All copyright.

et al. 2015), i.e. those belonging belonging i.e. those 2015), Accepted Article phenotypic traits – body area (Fig. 7A), testes area (Fig. 7B), ovary area (Fig. 7C), total egg – on ISH (not shown). Intotal we observed 9cases where one of the five measured knockdown partial implying staining reduced RNA815_54 and of RNA815_26815.1 case the transcripts were successfully knocked down, as determined by the absence of staining – or in (using the same methods described as above) on the treated and control worms: 7 out of 12 We verified whether knockdownwas successful by conductingsubsequent a ISH experiment (Lengerer expected substrate—was a to adhere to inability phenotype—the RNAi clear a which for and system, reproductive the transcript, macif1, that functions in the anchor cells of the adhesive system, rather than in theto relevant dish.As apositive control,we also performed RNAi anotheron tail-specific added was probe dsRNA no that except treatments RNAi the to identically treated was that MLRNA110815_26815.1).negativea As control, included we an additional treatment group tranpattern reverse exhibiting(testisthe two with the MLRNA110815_35903), MLRNA110815_1085, MLRNA110815_10574, remaining MLRNA110815_54, MLRNA110815_3206.2, MLRNA110815_13829.2, MLRNA110815_24606)ovary or (down in octets:MLRNA110815_1214.1, inthe inpattern (up testis octets: MLRNA110815_14701, MLRNA110815_21744, testis- and ovary-specific candidates. Specifically, most transcripts exhibited the prevailing gametogenesis pathways, selecting 12 transcripts from among the differentially expressed differentially expressedcandidates identifiedwe have indeed are likelytoin play roles RNAi screen putativeof testis- and ovary-specific transcripts to confirm that the many await the futurefollow-upoutcome of experiments,we performedsmall-scale a prospective Although a comprehensive functional picture is beyond the scope thisof study and must confirms roles RNAi for differentially expressed candidates in reproductive pathways This article by article rightsprotected This reserved. is All copyright. treatment and the control. For body area, MLRNA110815_14701 knockdown worms were number (Fig. 7D) or received sperm score (Fig. 7E) – differed between a specific RNAi et al. et 2014). scripts down in octets: MLRNA110815_10265,

Accepted Article MLRNA110815_14701 ( MLRNA110815_14701 both MLRNA110815_14701 (Fisher Exact Test: Test: Exact (Fisher MLRNA110815_14701 both ( developing than controls eggs more contained had significantly larger ovaries than controls. MLRNA110815_1214.1 knockdown worms ( LSD = 249, significantlywere larger controlsthan ( comparisons: significantly smaller than controls (ANOVA, Dunnett’s posthoc test for multiple-to-one This article by article rightsprotected This reserved. is All copyright. lateral bristles and a terminal brush; Ladurner Ladurner brush; terminal a and bristles lateral morphology as the sperm in control worms (an anterior feeler, aposterior shaft, a pair of with received sperm; mature sperm released from the testis exhibited the same general observed worms few very and 7G), (Fig. sperm of accumulation an containing apparently expression, Fig.6C) involved smaller bodysize (Fig.7F), markedlylarger testes size MLRNA110815_14701RNAi phenotype (whichwe had selectedbased testis-specific on To illustrate and expand on these observations with respect to specific transcripts, the observedfor the MLRNA110815_21744 RNAi). resultspecificthe of knockdown (the exception wasthe larger than expected body size knockdown was successful, strongly suggesting that the phenotypes we observed were a these9 effectsout of occurred amongthe transcriptsseven wherewe could confirmthatthe wefound0.00002), a smaller proportion of worm more more eggs (Fig. 7I). canAs beseen in Figs. 7J-L, the MLRNA110815_10574 and completea absence wormsof observed withreceived sperm, moderately larger testes and basis of specific expression in ovaries and developing eggs, Fig. 3W), knockdown resulted in wereand most partiallycompletely or immobile. For MLRNA110815_1214.1 theon (selected contrast to controls, a high number of sperm showed a marked bend in the shaft (Fig. 7H) ∣ diff ∣ – LSD = 250, = 250, –LSD P = 0.02) had significantly larger testes than controls. Both MLRNA110815_54 MLRNA110815_54 Both controls. than testes larger significantly had =0.02) ∣ diff ∣ – LSD = 7239, 7239, –LSD = P = 0.007) and MLRNA110815_10574 ( MLRNA110815_10574 and 0.007) = ∣ diff ∣ – LSD = 7262, 7262, = – LSD P = 0.009) MLRNA110815_21744 and knockdown worms ∣ diff ∣ – LSD = 6997, P < 0.0001) MLRNA110815_1214.1 and ( et al. ∣ P diff = 0.0004) and MLRNA110815_1214.1 ( s with received with in sperm 8 s their antrum. ∣ 2005b; Vizoso Vizoso 2005b; – – =LSD 0.054,

P ∣ diff = 0.0095). Both ∣ – LSD =389.3, et al. P = 0.03). Finally, for 2010),but in P = 0.002) = ∣ diff P ∣ – = Accepted Article have gained with respect tosex allocation in hermaphrodites and compare these results to we insights specific the of some explore then patterns, gene expression upon impact strongly conditions social that finding general the discuss first we following, the In allocation. of investment reproductive balance into the male and female functions, sex sex i.e. theaffecting overall spermcompetitionlevelof experienced andthus affectingoptimalthe with conspecifics on interactions is based response the case, this In itself. finds organism optimal gene expression pattern depends upon the particular ecological context in which an ifthe as expected is precisely This tissues. reproductive in specific uniquely expressed sensitive in its expression, especially with respect tothe subset of the transcriptome of the proportion a large that demonstrate results Our Discussion manipulate expression of these transcripts and thereby test their functions experimentally. transcripts with specific functions in reproductive tissues. They also illustrate our ability to screen has identified plastica subset of the identified throughour RNA-Seq ISHand experiments, clearly and demonstratethat our Collectively,RNAi these results correspond the to well expected expressionpatterns al. exhibited the usual non-adhesive phenotype associated with macif1 knockdown (Lengerer hadlarger markedly comparedovaries controlto worms. expected, As control worms expression, withMLRNA110815_54also being expressed in developing eggs(Fig. 3S,Y) – MLRNA110815_54 knockdowns – both of which were selected based on ovary-specific This article by article rightsprotected This reserved. is All copyright. sexes. separate with species in expression transcript sex-specific of patterns 2014). M. lignano transcriptome that includes includes that transcriptome M. lignano

transcriptome is socially- is transcriptome et Accepted Article impacts upontheexpression genes of that are likely differentiallyto affectthe or one other disproportionately environment social the altering that evidence strong provide data Our function-specificeffects Sex expression(Slavich Cole 2013).& gene human on influence significant a are factors social that recognized increasingly is expressionoccur during sex change in sequential hermaphrodites (Liu (Todd inspermatogenesis invest differentially to males different for pay often it may as context, in this interesting particularly gonads of fish following changes in social rank or adopting different morphs are also alternative castes (Sumner (Sumner castes alternative (Fraser tactics reproductive male alternative with in triggering wholesale changes in gene expr role major a play also can environment social The interaction. treatment × sex significant a status). Note, however, that this figure does not include the class of genes (1.4%) that exhibit expressed in different social environments (larval crowding, adult crowding or mating aspects of the environment, butaround 0.25-1.34% genomeof the was differentially chemical or nutritional physical, manipulate to sought treatments these of majority The 8% theof transcriptome (1249/14400 transcripts investigated) is plastic in its expression. todesigned manipulate different aspects environment. ofthe found They that overall around measuring gene expression in whole flies raised in oneof 20 different treatment groups performed a microarray-based study of how expression varies in varies expression ofhow study a microarray-based performed conditions,predominantly in separate-sexedtaxa. For example,Zhou al.et (2012) environmental social to responses genome-wide investigated have that studies previous to To put the global patterns of plastic gene expression in context, we comparecan our results geneexpressionof plasticity Global patterns This article by article rightsprotected This reserved. is All copyright. et al. 2006). The changes in gene expression that occur in the et al. 2018). And even more radical alternations in gene ession underlying alternative morphs in taxa intaxa morphs alternative underlying ession et al. 2014), or in social insects with

D. melanogaster et al. 2015). Finally, it , based on on , based Accepted Article antagonism (reviewed in Wilkinson identifygenesthe underlying sexualdimorphism and shaped sexualby selectionsexual and methods to document sexdifferences in geneexpression in separate-sexedtaxa, inorderto Our results can be compared to the increasing number of studies that have used similar with conserved function.consistent a reproductive (Weber region pharynx inthe also expression additional with although prostate, and (weakly) testis the both in patterns expression similar highly exhibit MLRNA110815_5404.3) and (MLRNA110815_5875.1 these of two least female reproductive tract interactions (Cho 2012; Choi surface membrane protein ADAM2 implicated in mammalian sperm-egg and/or sperm- the tometallo-peptidasebelong family reprolysin-like,M12B includeswhich sperm the differentiationin mouse (Saade (MLRNA110815_3206.3)family SPATIAL spermatidprotein to linked tothe matched potential reproductive functions. For example, one putatively gonad-specific transcript Haerty expected for genes sex-specificwith functions (Begun & Lindfors 2005; Begun transcripts are likely to be phylogenetically restricted or lineage-specific, as might often be to only two further melanogaster exhibit significant homology to humans or three model organisms (i.e. the fruit fly not did candidates 100 plastic top the of 38 Moreover, candidates. orovary-specific testis- exhibit76% prostate-limitedexpression; Weber and expression, prostate-specific confirmed have which of (90% candidates tail-specific are these of half fully 2: Fig. in illustrated transcripts plastic most 100 the among classes these found for the putative testis-, ovary- and tail-specific classes, as well as the dominance of sex function in This article by article rightsprotected This reserved. is All copyright. et al. 2007). Among those that did match to other taxa, there are also some hints at , the nematode, the M. lignano S. mediterranea (Fig. 3). This is clear from the patterns of differential expression C. elegans et al. et . This is consistent with the idea that many plastic et al. 2007), and several putatively tail-specific candidates or the triclad flatworm 2015; Grath & Parsch 2016). This is of course a et al. et et al. et 2018), which is at least partially 2018)), and a further 15 are either et al.

2016). Wenow know that at S. mediterranea et al. et ), and a D. 2007; Accepted Article to their intermediate, sexually dimorphic phenotype (Pointer (Pointer phenotype dimorphic sexually intermediate, their to corresponding genes, female-biased for ‘feminized’ and genes for male-biased masculinized’ gallopavo to reduced sexual antagonism (Hollis due potentially patterns, expression gene male ‘feminizing’ dimorphism, sexual reduced for (Ranz much higher proportion of genes exhibit some degree of sex bias in their expression level a though 2010), Morrow & (Innocenti females or males either in expressed when selection studies in (Schärer returns fitness their net to maximise so as functions sex female the into and male investment true sexual dimorphism,individuals and instead somewhat different question, since in simultaneous hermaphrodites there is no possibility of This article by article rightsprotected This reserved. is All copyright. melanogaster in environments socio-sexual different to responses expression transcriptome-wide organisms – at –sexed least recent is of interms a of exploration male investment Schärer hermaphrodites equates toantagonisticpleiotropy) (AbbottJordan 2010;Connallon & 2014; expression optimum differs between the sexes or sex functions (which in simultaneous between the two states can be thought of as being sexually antagonistic, in that their expression in differing genes of subset the cases both In allocation. sex female-biased more or male- more favouring environments different in expression gene shift plastically or social status, we asked instead how the same (hermaphroditic) individual might In this study, rather than considering fixed candidate genes implicated in the highly plastic responses of male flies to perceived changes changes perceived to flies of male responses plastic highly inthe implicated genes candidate identify to rivals, to exposure of periods time various after males focal by responses et al. et et al. et al. ), compared), todominant malesthe gene expression subordinate of is ‘de- males D. melanogaster 2003). Moreover, it has been suggested that the imposition of monogamy selects selects monogamy of imposition the that suggested been has it Moreover, 2003). 2015). Finally, perhaps the most directly comparable study to ours in separate- in ours to study comparable directly most the perhaps Finally, 2015). 2015; Schärer &Ramm 2016). Nevertheless, thereclear are parallels. Recent (Mohorianu (Mohorianu found that 8% of the genome experiences genome foundthat sexually8% ofthe antagonistic et al. 2017). This study sought to measure gene expression gene expression measure to sought study This 2017). et al. 2014). Similarly, in wild turkeys ( turkeys in wild Similarly, 2014). differences between individuals differing in sex face an optimisation challenge of balancing

et al. 2013). Meleagris D. Accepted Article

& Schärer 2014; Ramm see Mank (n transcripts candidate specific transcripts overall there means that arefive-fold at least more socially-sensitivetestis- differentially expressed in these two tissues, farthe greater number of testis-specific ovary-specific transcripts in our dataset. Although a similar proportion of transcripts are over testis- expressed of differentially the preponderance is observation interesting Another aspectsof ejaculate production,i.e. and seminal sperm fluid. in both implicated genes of expression in differences marked and in broad-scale results demonstrating asustainedthat exposure todifferentspermcompetition in levels between resu consistent Our replicates. always sperm) genes changed markedly in expression between treatments, although this was not of ejaculate components. Nevertheless, the authors noted that several seminal fluid (but not production than rather allocation of level the at primarily is circumstances these under flies overall much more stable in expression, which could imply that the plasticity observed in was region abdomen the contrast, By cues. sensory multiple to responding are males that idea the supporting observed, were region head/thorax the in in expression changes initial Substantial respectively. systems, reproductive and nervous inthe occurring responses to tocorrespond expected abdomen, and head/thorax the regions, body two to according in immediate sperm competition risk and intensity. They further split these responses This article by article rightsprotected This reserved. is All copyright. example, in sex-specific genes appears to common be a ph n plus inclass [0,+,0] 125 et al. D. melanogaster 201) for an opposite trend in chickens; reviewed in Kaessmann 2010; Ramm Ramm inKaessmann 2010; reviewed inchickens; trend for an 201) opposite et al. ≤ 80 in class [+,+,0]). This apparent male-bias in the number of 2014). and ≤ C. elegans 1,053) than ovary-specific transcripts (n (Reinke enomenon, with similar evidence found, for lts clearly confirm and extend these findings, extend and these confirm lts clearly et al.

2000; Chintapalli ≤ 205; i.e. n et al. M. lignano 2007; but ≤

Accepted Article Mohorianu Mohorianu exhibit such plasticityin for demand fluid.seminal A small number investigated of seminal fluid genes and proteins fluid genes driven by variation in the socio-sexual environment, which likely modulates the supporting studies in separate-sexed taxa indicating plasticity in the expression of seminal plasticity reported to date (but see Hollis (see also(Patlar highlight the scope also for considerable intraspecific plasticity in seminal fluid expression al. (Dorus levels competition insperm variation inter-population or inter-specific to linked Moreover, the rapid evolution and divergent expression of seminal fluid proteins beenhas 2012). Fitzpatrick & (Simmons investment reproductive male to approach integrative an of well recognised in a range of taxa (Poiani Avila2006; function and evolutionary significance of seminal fluid-mediated fitness traits is increasingly The allocation. male of aspects non-sperm of importance likely the emphasizes further Given our clear results for seminal fluid as well as putatively testis-specific genes, our study This article by article rightsprotected This reserved. is All copyright. et al. 1998; & 1979; Michiels Koene Michiels (Charnov 2006; Marie-Orleach and for which unique functional hypotheses about seminal fluid have been proposed important, especially be to expected are fluid, seminal by mediated those as such processes, simultaneously hermaphroditic modelsystem, in which post-copulatory sexuallyselected data now provide a rich repertoire of candidates for probing seminal fluid function in a 2017; Sloan Lovegrove & (Simmons intensity and risk competition sperm Teleogryllus oceanicus proteome differs according sperm to competition risk(Ramm responses can be highly variable), in mice, the relative composition of the seminal fluid 2004; Ramm 2014; Schärer et al. et al. 2017 – discussed above – for evidence that seminal fluid expression expression fluid seminal that evidence for – above discussed – 2017 et al. et al. 2018)). This is, to our knowledge, the largest scale evidence of such 2008; 2009; Goenaga , males adjust seminal fluid production in response to cues of both both of cues to inresponse production fluid seminal adjust , males Drosophila 2015). (Fedorka (Fedorka et al. et al. 2016 for a similar evolutionary response), response), evolutionary similar a for 2016 et al. 2015; Hollis 2011; Wigby et al.

2011), stressing the importance importance the 2011), stressing et al. et et al. et al. 2015), and in the cricket inthe cricket and 2015), 2016).Our results 2015) (but see et al. et et al. et 2013; Nakadera 2018). Our et Accepted Article larger groupsize, in line with predictions fromsex allocation theory (Charnov 1979; 1982). at allocation sex male-biased more a towards size group small at allocation sex female-biased more a from investment their shift worms as conditions, social prevailing the to according the of proportion substantial a that evolutionary significance (Aubin-Horth & Renn 2009; Alvarez now studied, for can traitsbe of reaction “genomic ecological norms” defining or sequencing in Advances technologies the molecular meanthat of phenotypicbasis plasticity andOutlook conclusions 2015). exhibits homology to ABCA8, which is enriched in both male and female gonads (Uhlén maleaccessory reproductive glands (prostate, seminalvesicle) and MLRNA110815_2216.1 MLRNA110815_18363.2 exhibit homology to the human gene WSCD2, which is enriched in and MLRNA110815_18363.1 interestingly but taxa, other to homology (absent) on based unannotated functionally remain also Several ofexpression. sites specific their theyexhibitthat sperm-limited expression,no thoughhavewe directdate evidence to on patterna could be that these transcripts are expressed only in male tissues, or possibly even MLRNA110815_5404.2, butnot MLRNA110815_4414) and MLRNA110815_471.1,MLRNA110815_324.2, MLRNA110815_471.2 MLRNA110815_18363.1,MLRNA110815_2216.1, MLRNA110815_18363.2, between expressed differentially transcripts this in [+,0,+]class,our analyses indicatethat fully eight theseareof 2015). Although, according the the testis region (i.e. B vs. A contrast) and the tail region (i.e. D vs. C contrast) (Arbore investigation is the small number of transcriptswhose positional expressionhighis inboth In the context of male allocation, another class of genes that might repay further This article by article rightsprotected This reserved. is All copyright. to Arbore et al. (Arbore (Arbore al. et Arbore to isolated isolated M. lignano and octet transcriptomedifferentially is expressed worms (MLRNA110815_14112, (MLRNA110815_14112, worms et al. et .One plausible interpretationsuch of 2015), there are only nine such

et al. et 2015). We have shown shown 2015). have We et al. et al.

Accepted Article such as ISH and RNAi are readily applicable in characterizationfrom our genome-wide scan,andhave demonstrated that moleculartools repert comprehensive a with equipped now towards a complete mechanistic understanding of the phenomenon. Nevertheless, we are response to sex allocation plasticity represents a necessary but still insufficient first step plasticity is achieved. Of course, understanding the genes exhibiting altered expression in allocation sex how into insights significant and provide species, this in expression gene These data establish the key role played by the biotic environment in generating variation in This article by article rightsprotected This reserved. is All copyright. phenotypic plasticity in sex allocation, a crucial life history trait. hermaphroditic animal, and thus begin to bridge organismal and molecular perspectives on favouring shifts in investment towards one sex function or the other in this model in environments expressed differentially that are genes the define results now, our For A. lyrata selection (based on patterns of intraspecific polymorphism and interspecific divergence from positive of incidence higher a show indeed tubes, pollen and pollen in expressed genes, hermaphroditic plant inthe evidence is some taxon. There animal hermaphroditic inthis genes specific sex- for also up holds sexes separate with species in genes sex-specific among evolution rapid al. rapid divergence of sex allocation and sexually selected traits in hermaphrodites (Schärer an excellent model taxon complementaryfor genomic and transcriptomic analyses of the genes. Moreover, the genus 2013), enabling tous now test the functional and evolutionary significance ofindividual

2011). It will, for example, be fascinating to examine whether the striking pattern of more ) (Gossmann (Gossmann ) Arabidopsis thaliana et al. 2014). Macrostomum

, comprising >150 described species, represents represents species, >150 described , comprising oire of candidate genes of functional candidate for oire suggesting that at least some male-specific male-specific some least at that suggesting M. lignano

(see alsoe.g. Sekii et al. 2009; et et Accepted Article manuscript. final the approved and read and drafted the manuscript: SAR, LS. All authors contributed to manuscript revisions, and data the PL. Analysed AG and JW, RP, BL, RNAi screens: and ISH the Performed EB. reads: Performedthe sex allocation assay: SAR,LS. Extracted RNA: RA.sequenced Mapped the Study conception and experimental design: SAR, LS. Performed the main experiment: SAR. Author contributions (https://www.ncbi.nlm.nih.gov/sra?term=SRP174213). deposited in the Sequence Read Archive under accession number SRP174213 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE123594). Raw data are also Omnibus and are accessible through GEOSeries accession number GSE123594 RNASeqdata derivedand read counts,has been depositedin NCBI'sGene Expression includingstudy, MLRNA110815associated the with the Data transcriptome assembly, accessibility Data PhD Fellowship of the University of Innsbruck. and the Austrian Science Fund (FWF) grant P 25404-B25 (to PL). BL was supported by a RA 2468/1-1 (to SAR); the Swiss National Scie CurieIntra-European Fellowship toSAR); th Funding was provided by the Seventh Framework Programme of the European Union (Marie Acknowledgments This article by article rightsprotected This reserved. is All copyright. e German Researche Foundation (DFG) grant nce Foundationgrant 31003A-143732 (to LS);

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, 323. Sperm Competition and and Competition Sperm . Animal Behaviour Animal Current Biology Journal of Evolutionary Evolutionary of Journal Current Biology Current , 27 Biology of Sex Sex of Biology , 1570– RNA (New , 14 , 85 , , 1509– 24 , , Accepted Article Ramm SA, Edward DA, Claydon AJ AJ DA,Claydon Edward SA, Ramm Ramm SA, Oliver PL, Ponting CP, Stockley P, Emes RD (2008) Sexual selection and the the and selection Sexual (2008) RD Emes P, Stockley CP, Ponting PL, Oliver SA, Ramm Ramm SA, McDonald L, Hurst JL, Beynon RJ, Stockley P (2009) Comparative proteomics Ramm SA, Schärer L (2014) The evolutionary ecology of testicular function: size isn't Schärer L (2009) Tests of sex allocation theory in simultaneously hermaphroditic animals. animals. hermaphroditic in simultaneously theory allocation sex of Tests (2009) L Schärer Saade M,M,Govin Irla J Rieger RM, Gehlen M, HaszprunarG Reinke V, Smith HE, Nance J Ranz JM, Castillo-Davis CI, Meiklejohn CD, Hartl DL (2003) Sex-dependent gene expression of evolution the and competition Sperm (2014) J Wistuba J, Ehmcke L, Schärer SA, Ramm is expression gene of Masculinization (2013) JE Mank AE, PW, Wright MA,Harrison Pointer This article by article rightsprotected This reserved. is All copyright. Schärer L, Vizoso D(2007) Phenotypic plasticity in sperm production rate: there's more to it Schärer L, Ramm SA (2016) Hermaphrodites. In: Schärer L, Janicke T (2009) Sex allocation and sexual conflict in simultaneously Schärer L,Janicke T, Ramm SA (2015) Sexualconflict inhermaphrodites. Schärer L, Ladurner P (2003) Phenotypically plastic adjustment of sex allocation in a 25 proteins. ejaculate mammalian of evolution adaptive 189–198. competition. in sperm significance functional reveals evidence for evolutionary diversification of rodent seminal fluid and its composition. fluid seminal everything. Evolution 313 Macrostomida (Turbellaria). elegans. transcriptome. Drosophila the of evolution and spermatogenesis. associatedwith exaggerationsexual ofmale dimorphism. Harbor PerspectivesHarbor inBiology thansize. testis 1–29. 270 animals. hermaphroditic spermatogenesisand its interaction withthe kinesin KIF17b. simultaneous hermaphrodite. hermaphrodite. simultaneous , 207–219. , 614–626. , 935–941.

Molecular Cell , 63 Biological Reviews , 1377–1405. Evolutionary Ecology Molecular Human Reproduction et al. , 6 Biology Letters et al. , 605–616. , 605–616. (2007)Dynamic distributionSpatial of during mouse BMC Biology BMC 36 Proceedings of the Royal Society B: Biological Sciences Biological B: Society Royal the of Proceedings (2000) Aglobal profile of germline gene expression in , , et al. , 523. , 523. 89 7 et al. , a017673. , a017673. , 874–888. , 874–888. (2015) Sperm competitionrisk drives plasticity in , (1988) Laboratory cultures of marine marine of cultures Laboratory (1988) 21 , , 13 , 295–306. , 295–306. 5 , 705–708. , 87. , 87. Encyclopedia of Evolutionary Biology Evolutionary of Encyclopedia Molecular Biology and Evolution and Biology Molecular Science , 20

Molecular Biology and Evolution , , 1169–1179. 300 PLoS genetics PLoS , 1742–1745. , 1742–1745. Experimental cell research cell Experimental Cold Spring , 9 , e1003697. , 26 , , pp. , pp. C. , , , Accepted Article Schärer L,Ladurner P, Rieger(2004b) RM Bigger testes work do more: experimental Schärer L, Joss G, Sandner P (2004a) Mating behaviour of the marine turbellarian This article by article rightsprotected This reserved. is All copyright. Schärer L,Littlewood DTJ, Waeschenbach A, YoshidaW, Vizoso (2011)DB Mating behavior Schärer Schärer L, P, Sandner Michiels (2005) N Trade-off between andmale female allocation in SchleicherovaLorenzi D, MC,SellaMichiels G, NK (2010) Gender expressionand group Schärer L, Zaubzer J, Salvenmoser W, Seifarth C, Ladurner P (2007) Tracking sperm of a Sekii Sekii K, Vizoso DB, Kuales G Sekii SalvenmoserK, MulderW, De K, LadurnerMelav2, P(2009) elav-like angene, is Sloan NS, Lovegrove M, Simmons LW (2018) Social manipulation of sperm competition mediates expression gene fluid seminal cued (2017) Socially M Lovegrove LW, Simmons fertility. male of evolution the and wars Sperm JL (2012) Fitzpatrick LW, Simmons Tan GN, Govedich FR, Burd M (2004) Social group size, potential sperm competition and and competition sperm potential size, group Social (2004) M Burd FR, Govedich GN, Tan phenotypic and expression gene Differential WC (2006) Jordan JJM, Pereboom S, Sumner Slavich GM, Cole SW (2013) The emerging field of human social genomics. invertebrate, the free-living flatworm evidence that testis size reflects testicular cell proliferation activity in the marine Macrostomum 108 design. sperm of evolution the and Sociobiology Evolutionary Biology the simultaneously hermaphroditic flatworm size: a test in a hermaphroditic and gonochorica congeneric species of donor in a recipient: an immunocytochemical approach. Royal Society B: Biological Sciences Biological B: Society Royal theory. competition sperm of predictions evolutionary confirms essential for spermatid differentiation in the flatworm (Polychaeta). intensity reduces seminal fluid gene expression. expression. gene fluid seminal reduces intensity 348. Sciences Biological B: London of Society risk. competition sperm to quality inejaculate responses Reproduction (Euhirudinea: Glossiphoniidae). Glossiphoniidae). (Euhirudinea: reproductiveinvestment hermaphroditicin a leech, Proceedings of the Royal Society B: Biological Sciences plasticity in behavioural castes of the primitively eusocial wasp, Science Psychological for Association the of journal a : science psychological , 1490–1495. , 1490–1495. , Journal of Experimental Biology Experimental of Journal , 56 sp.: these worms suck. 144 , 420–425. , 519–534. , 18 et al. , 396–404. (2013) Phenotypic engineering of sperm-production rate Journal of Evolutionary Biology Evolutionary of Journal Proceedings of National the AcademySciences of , Macrostomum 280 Marine Biology , 284 . Macrostomum , 20171486–8. , 20171486–8. , 213 Biology Letters Biology Helobdella papillornata , 1586–1590. , 1586–1590.

sp. sp. , Macrostomum lignano 145 , Behavioral Ecology and and Ecology Behavioral Animal Biol. 273 Proceedings of the Royal , 373–380. sp. , 19–26. , 19–26. , Polistes canadensis Proceedings ofthe Journal of 14 , 17 , 20170659–4. , 20170659–4. , 574–580. , 57 Ophryotrocha Clinical , 121–136. , 121–136. , . 1 9 , 331– , 62. , 62. . ,

Accepted Article Zhou S, Campbell TG, Stone EA, Mackay TFC, Anholt RRH (2012) Phenotypic plasticity of Yampolsky LY, Zeng E, Lopez J Wudarski J, Simanov D, Ustyantsev K Wilkinson GS, Breden F, Mank JE Wigby S, Perry JC, Kim YH, Sirot LK (2015) Developmental environment mediates male male mediates environment Developmental LK (2015) YH, Sirot Kim JC, Perry S, Wigby The (2000) allocating sex. of Sheldon benefits SA,Herre BC EA, West SA(2009) West Weber M,Wunderer J,Lengerer B Wasik K,Gurtowski Zhou J, X VizosoRieger D, G, SchärerL (2010) Goings-on insideworm: a functional hypotheses Uhlén M, Fagerberg L, Hallström BM Trouvé S, Jourdane J, Renaud F, Durand P, Morand S(1999) Adaptive sex allocation in a Todd EV, Liu H, Lamm MS This article by article rightsprotected This reserved. is All copyright.

the adaptation in response to thermal stress in Communications sequencegenome ofthe regenerative flatworm Macrostomummodel lignano. era. post-genomics the into studies selection 419. seminal protein investment in 290. Biology Evolutionary BMC flatworm. hermaphroditic in simultaneously a proteins fluid seminal putative identifies Sciences competent flatworm, 370–383. derived from sexual conflict thinking. human proteome. proteome. human hermaphrodite. simultaneous Molecular Biology and Evolution Transcriptomic Signature in Both the Brain and the Gonad in a Sex-Changing Fish. Drosophila , 112 Sex Allocation Sex , 12462–12467. transcriptome (GS Barsh, Ed,). , 8 Science , 1–12. , 1–12. Macrostomum lignano et al. , et al. . Princeton University Press. 347 , et al. 18 (2018) Female Mimicry by Sneaker Males Has Has a Males Sneaker by Female Mimicry (2018)

Evolution et al. Drosophila melanogaster et al. , 1260419. , 1260419. , 81. , 81. (2015) Genome and transcriptome of the regeneration- of the transcriptome and Genome (2015) (2014) Functional genomics of acclimation and , et al. et al. 35 (2015) The locus of sexual selection: moving sexual (2018) A targeted in situ hybridization screen screen hybridization insitu A targeted (2018) , 225–241. , 225–241. Biological Journal of the Linnean Society (2015) Proteomics. Tissue-based map of the (2017) Efficient transgenesis and annotatedand transgenesis Efficient (2017) , 53 Daphnia , 1599. J. Evol. Biol. Evol. J. . Proceedings of the National Academy of of Academy National the of Proceedings PLoS Genetics .

BMC Genomics BMC . , Functional Ecology 28 , , 739–755. , 739–755. 8 , e1002593. , e1002593. Science , 15 , 859. , 859. , 290 , Nature 30 , 288– , 99 , 410- , Accepted Article transcripts differentially expressed accordingto‘mating groupsize’; (D)that and subset a of transcriptomeof thesubset is differentially expressedbetween worms, i.e. transcripts differentially expressed when ‘not mating vs. mating’; (C) that a transcriptome corresponding to three specific biological hypotheses: (B) that a subset of the the of subset a that (B) hypotheses: biological specific three to corresponding transcriptome do differ from one another. We used these estimates to address the proportion of the same border do not differ from each other, whereas those surrounded by different borders specificdifferential type of expression each classrepresents: treatments surrounded the by classes, depictedseparately belowchart highlighting pie and the with dotted borders the transcripts, as depicted in the pie chart). Next, we examined the estimates from relevant belonging to one of the seven different differential expression classes (ca. 19% of all posterior probabilities for all transcripts toestimatethenumbertotal transcripts of summed then We another). one from differ not did groups treatment four all which in class groupdiffered in expressionlevel comparedothers, tothe ‘notplus a differentially expressed’ expression classes (seven biologically plausible patterns in which leastat one treatment estimated the likelihood that each transcript belonged to one of eight different differential differential expression patterns in the of impression global a gain to transcripts all from information incorporating by began We environment in Figure 3.Global patterns of differentialexpression gene according the social to assemblies. between converting with Suppl. Tables S3 and S4, which provide corresponding transcript IDs for inter- in combination 2018), al. et Grudniewska 2017, al. et (Wudarski assembly transcriptome all remaining transcripts please see those provided for the Mlig_RNA_3_7_DV1 For full functional annotations, see Suppl. File S5, for and similar functional annotations of the identitytopthe of (lowest E-value) homologhuman scoringand top Pfam assignment. melanogaster identified homologs in humans (Hm) and three model organisms ( M. lignano in patterns expression positional their including information functional of a summary with replicateisolated treatments (blue) andfourreplicate octet environments (red),together For each of the top 100 plastic candidates, we plot their expression patterns in the four environment in plastic candidate transcripts differing in expression accordinggene the to social Figure 2.Per replicate expression andpatterns functional annotations for the top 100 perspective. landscape transcriptional now well-established phenotypically plastic response that we here investigate further from a by a represents This details). for text main (see treatment size group the with significantly allocation (astestis area (testis / + ovary area area)). Weconfirmedthat sex allocation varies these transparent flatworms (as illustrated in the inset) and used to derive a proxy for sex investmentThe into testes (Te,blue)and ovaries (Ov,orange) can bereadily quantifiedin perspective. Figure 1.Sexallocation plasticity in Figure Captions This article by article rightsprotected This reserved. is All copyright. transcriptome is differentially expressed between (Arbore et 2015),al. the presence (filledsquares) or absence (open squares) of

, Dm; , Dm; Macrostomum lignano Macrostomum lignano Caenorhabditis elegans M. lignano Macrostomum lignano Macrostomum . . , Ce; , Ce; Schmidtea mediterranea Schmidtea transcriptome. (A) Using isolated

and grouped ( pair from a morphological Drosophila and octet pair , Sm) wellas as baySeq and worms, i.e. i.e. worms, octet , we , we ) Accepted Article through the pairwise pairwise the through candidates. Each scatterplot is based on differentially expressed candidates identified (orange), ovary-specific (E) [+,+,0](magenta) and (F)tail-specific [0,0,+](green) non-specific [0,0,0] (red), (C) testis-specific [+,0,0] (blue), (D) ovary-specific [0,+,0] each of these positional classes are plotted separately in colour in to thebelonging subsequentcandidates expressed Differentially panels. panels: subsequent in worms of drawings (B) line inset smaller inthe indicated as expression, tail-specific or ovary-specific testis-specific, non-specific, either have to expected candidates putative as analysed transcripts 76,437 log between key reproductive traits (Arbore generated data has been frominto worms cut fragments corresponding theto boundaries stylet the is the copulatorymale organ.Owingto their transparency,previous transcriptomic sperm-receiving female the organ, seminalvesicle theisstoragesperm the male organ and is antrum The 1.5mm). ca. length body (total features morphological key indicating 2007)), for reference (bold black symbols and lines).The numbersrefer to the average changesin plotted in black. The mean expression level in each of the three treatments is superimposed expression in octets, and 10 tail-specific candidates with lower expression in octets) are higher with candidates ovary-specific 5 in octets, expression lower with candidates differed in expression in the opposite-to-prevailing direction (i.e. testis-specific 20 that transcripts for norms reaction that except 3, Figure to corresponding colours in plotted the quantitative nature of sex allocation plasticity. The raw data for each transcript are of class [0,0,+](N =150, isolated-pairs: 2.240.30; ± pairs-octets: 0.59 ± 0.30), illustrating 80, isolated-pairs: -0.62 ± 0.36; pairs-octets: -0.49 ± 0.36) and (D) tail-specific candidates 0.55 ± 0.29; pairs-octets: -0.51 ± 0.29), (C) ovary-specific candidates of class [+,+,0] (N = octets: 0.35 ± 0.08), (B) ovary-specific candidates classof [0,+,0] (N = 125, isolated-pairs: - = 1,053; mean ± SE slope connecting adjacent treatments, isolated-pairs: 0.63 ± 0.08; pairs- Reaction norms for differentially expressed (A) testis-specific candidates of class [+,0,0] (N allocation in of sex gametogenesisTranscriptomic aspects andother for norms 5. reaction Figure reference. for conditions, that positional class. In each plot (B-F), the red line indicates equal expression in both social differentially expressedcandidates compared of number to the relative total the number indicates of transcripts figure percentage the belongingthat below and to respectively, number the of that exhibithigher (up arrow)loweror (down arrow) expressionin panelrefer total tothe numberdifferentially of expressed candidates identified,thesum as expression data for all other transcripts (in grey). The numbers at the bottom right of each 0.10)thatfor positional class (in the correspondingcolour) plotted are abackground on of equivalent data for worms in (A) Photomicrograph and line drawing of the model organism (adapted from (Schärer (Schärer from (adapted organism model the of drawing line and Photomicrograph (A) the transcriptome is differentially expressed between This article by article rightsprotected This reserved. is All copyright. lignano TheFigure 4. transcriptionallandscape allocation of sex in plasticity proportion of the transcriptome to which this scenario could apply (stated below). statedinthe scenario (as highlightedinblue and red) toarrive atthe estimatedtotal each scenario (B-D), wesummed differentialall expression classes that fulfil the criterion transcripts differentially expressed‘e an as 2 -transformed expression level for four biologically independent replicates) against the .

M. lignano M. DESeq . comparisonof expression in octets ( y axis). Differentially expressed candidates (all FDR < et al. arly arly response’ when commencing mating. For 2015), thereby enabling us here to assign the isolated

isolated and joined worms ( worms, i.e. i.e. worms, x Macrostomum axis, mean of octets et al. ,

Accepted Article ag vre O) large ovaries (Ov). MLRNA110815_54 (K) and MLRNA110815_10574 (L) knockdowns can be observed to have present, each highlighted with an arrowhead (I). Compared to controls (J), both illustrate worms knockdown the MLRNA110815_1214.1 number large typically of eggs region,indicated as the by arrowheads (H).Representative images for four accompanied by an unusual sperm morphology, in which many sperm were bent in the shaft body size enlargedand testes (Te) in MLRNA110815_14701knockdown worms (F,G) was significant treatment effects illustrated are right-hand onthe figure.Theof the side smaller panels (A-D),significant these effects are highlightedin red, in (E)byasterisks. Somethe of in for details): text (see main control the from macif1 significantly differed phenotypes antrum). In total, wefound nine instances where one of the testis/ovary RNAi knockdown (E)eggs; and receivedsperm status(i.e., presencetheor absence receivedof sperm the in suiteof traits: (A) body area; testes(B) area; (C) ovaries area;(D) number of developing examining worms under differential interference microscopy and measuring astandardized system (macif1) (Lengerer to be known control transcript expressed innon-reproductive the exclusively adhesive using expression ovary-specific or testis- have to confirmed We knockedthedown expression twelvedifferentially of expressed transcripts that wehad functions in gametogenesis pathways. reveals selectedtestis- andovary-specific transcripts of RNAi knockdown 7. Figure two are expressed in both gonads (Q, R). sensitive in their expression, oneis expressed in the ovary and in developing eggs (P) and the testis or at the testis periphery (M, O, T). Of the three candidates that are not sociallyregion) whereas for the other three, in addition to the ovary, there is also weak staining in inU,W, Xalso butnote X and single Y; S,V,small staining dots the in pharynx/anteriorgut &S-Y),M-O indeedseven exhibit expression limitedovary tothe developingand/or (N,eggs from classes [0,+,0] and [+,+,0]. Of the ten transcripts expected to be ovary-specific (panels and/or prostate (Pr).(L) Thirteen putatively ovary-specifictranscripts were investigated (Ph), postpharyngeal glands (Po), gut (Gu), ovaries, developing eggs (De), antrum (An) pharynx (Ro), the rostrum including also instead the to testis, never limited but including expression between isolated and octets (J-K transcripts that had,respectively, lower expression in octets (F-I),thator not diddiffer in staining in both testes and (perhaps more weakly) ovaries (Ov). By contrast, the four and two expression in octets (B-D) are testis-specific (Te) as predicted, while (E) exhibits ISH 3). All but one (I) fit the expected pattern. Three of the four transcripts with higher (A) Ten putatively testis-specific transcripts in class [+,0,0] were investigated (plot as in Fig. expression. expressed examiningtranscripts, putative those testis-or ovary-specificwith Figure 6. belonging to that positional class (see main text for details). expression between adjacent treatments, based on differentially all expressed transcripts This article by article rightsprotected This reserved. is All copyright. In situ In screenhybridization for tissue-specific expression differentially of et al. 2014).thenWe assessed reproductive phenotypes by ), exhibit varying ISH patterns, sometimes

in situ hybridization, plus one one plus hybridization, Accepted Article 2. in Fig. presented on data expanding transcripts, plastic top 100 the for transcriptome assembly Mlig_RNA_3_7_DV1_v3 the from annotations Full functional SupplementaryS5 File in transcripts transcriptome assembly.corresponding the MLRNA110815 assembly in Mlig_RNA_3_7_DV1_v3 all transcriptome and their the transcripts of List Supplementary File S4 assemblies. transcriptome Mlig_RNA_3_7_DV1_v3 corresponding MLRNA150904 thetranscript IDs transcripts studied, plus for and SupplementaryS1. Figure This article by article rightsprotected This reserved. is All copyright. Count data, treatment means and differential expression analyses for analyses and all 76,437 for data,differential Count expression treatment means SupplementaryS3 File primers of used List experiments. for the ISH and RNAi SupplementaryS2 File experiment. group size initial the following allocation plasticity sex for totest collected data Morphological SupplementaryS1 File the data a for of therein) (see in metadata tabcontained description full file each Supplementary Files et al. gonads or prostate), emphasizing that the earlier positional RNA-Seq classification (Arbore both eggs, developing ovary, (testis, expression reproduction-specific have fact in transcripts ‘non-specific’ these of proportion substantial a that note Also tissue. one than more for found greater than the number of transcripts, because for some transcripts specific expression was (Lengerer cells weak expression overall and/or in the gonad) and MLRNA110815_2945.1 topostpharyngeal exhibit to appearing also worms several 1 (with type glands frontal in expression weak to MLRNA110815_5085 likely corresponds to fron experiments, so the assignment is less certain. The staining in the pharynx region for (not shown).MLRNA110815_21158.2 exhibiteddifferent patterns twodifferent ISH in additional transcript (MLRNA110815_21158.1) exhibited an unspecific ISH staining pattern epidermis (ep), adhesive system (ad), cement glands (ce), testes (te) and rhabdites (rh). One (pr), prostate (ro), rostrum (ph), glands or pharyngeal region pharynx (ov), ovaries specific expression in tissues including the gut (gu), both gonads (go), developing eggs (de), The transcripts exhibited awide variety of ISH patterns (summarised in the inset), with differentially expressed, non-specific candidates. 2015) does not provide an exhaustive list of tissue-specific candidates. candidates. tissue-specific of list exhaustive an provide not does 2015)

et al. 2016). Note that the total sum of expression types shown in the inset is is inset the in shown types expression of sum total the that Note 2016). In situ In hybridization expression forpatterns 41 tal glands type 3, in MLRNA110815_26830 MLRNA110815_26830 in 3, type glands tal

M. lignano

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A B C D E Testis-specific candidates J Ro [+,0,0] Ph

Te Te Te Te Te Ov

K DJ RNA815_21744 RNA815_14701 RNA815_24606 RNA815_36754 RNA815_5848.1 C B H Higher expression in Octets E K F G H I G Gu F I Ph Ph Te Po Te Te Gu Te Ov Ov Ov De De An De RNA815_36731 Pr RNA815_26815.1 RNA815_20151 RNA815_7955.1 RNA815_10265 Not differentially expressed Lower expression in Octets L Ovary-specific candidates M N O P [0,+,0] and [+,+,0]

Te Te Ov Ov Ov Ov De De

R Q X P Y RNA815_19305 RNA815_1085 RNA815_35903 RNA815_9521.2 O W UV Lower expression in Octets [0,+,0] M N Q T S S T U Te Ov

Te Ov Ov Ov De RNA815_21095

RNA815_10574 RNA815_29287 RNA815_3206.2 R V W X Y Te Ov De Ov Ov Ov Ov De De De De RNA815_10777.2

RNA815_13829.2 RNA815_1214.1 RNA815_16539 RNA815_54 Not differentially expressed Lower expression in Octets [+,+,0] Accepted Article This article by article rightsprotected This reserved. is All copyright.