Posted on Authorea 2 Aug 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.162790498.82247747/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. igPn Xie Qing-Ping of gonochorism evolution and the hermaphroditism into insights croaker provide yellow polyactis) little (Larimichthys of annotation and assembly Whole-genome iiLiang Qiqi ieisgt noteeouino emprdts n gonochorism Liang and Xie hermaphroditism of Qing-Ping ( evolution croaker the yellow into little insights of vide annotation and underpinnings assembly genetic explained aquaculture. Whole-genome the be fish into may enhancing insight polyactis provides for only L. information not valuable male genome provides in polyactis also duplication stage L. but lineage-specific hermaphrodite the evolution a the Decoding from rnf225, hermaphrodite that dmrt1. originated of and shows regulatory of it rnf223 analysis evolution upstream that paralogues Phylogenetic sequence suggesting its two the teleosts. species, and its by other teleost dmrt1 unlike in other Rnf183, of in lost expression not was analysis. but The or transcriptome Larimichthys the in polyactis. in present L. dimorphic only gene in is dmrt1 sexually gene The both genes. determination were species. protein-coding two sex rnf183 25,233 the candidate gene between and within-chromosome events the of Mb) fusion as number 4.52 or a identified fission = revealed years origin chromosome was N50 which million major the ˜25.4 analysis, scaffold without sequence genomic crocea and Asia, translocations Larimichthys comparative genome Mb and of East enabled rearrangements the ancestor 1.21 assembly in common genome = report the high-quality species N50 we from Our (contig fish diverged Here, ago. polyactis Mb commercial L. ˜706 unclear. famous that of remain suggests most studying size analysis crocea Phylogenomic a for the Larimichthys with model as and polyactis, appealing hand, polyactis transient L. L. an other special of of polyactis) The the relationship L. On solved. evolutionary polyactis, be and (Larimichthys hermaphrodites. to croaker of mystery yellow intriguing formation little an the the is makes hermaphroditism stage and gonochorism hermaphroditic of direction evolutionary The Abstract 2021 2, August 4 3 2 1 .TeHdo lh nttt o itcnlg,Hnsil,Aaaa386 USA; 35806, Alabama Huntsville, Biotechnology, for Institute Alpha USA; Hudson 36849, 36849, The AL AL 4. Auburn, Station, Auburn, Experiment University, Agricultural Auburn Alabama Medicine, 3. Veterinary of College China; Pathobiology, 310021, USA; Hangzhou of Sciences, Department Agricultural of 2. Academy Zhejiang Hydrobiology, of Institute 1. uunUniversity Auburn University Xiamen available not Sciences Affiliation Agricultural of Academy Zhejiang 6 u Xie Yue , 2 u Xie Yue , 1# 6 1 egXu Peng , e Zhan Wei , e Zhan Wei , 2 egXu Peng , 5 ,X Wang Xu *, 1# 1 inZiShi Jian-Zhi , inziShi Jian-zhi , 3 uWang Xu , 2,3,4 ,BoLou Bao *, 6# 2 egLiu Feng , egLiu Feng , 4 n a Lou Bao and , 1 1 * 1 a-ogNiu Bao-Long , 1 a-ogNiu Bao-Long , 1 aiihhspolyactis Larimichthys 1 u He Xue , 1 u He Xue , 1 egLiu Meng , 1 egLiu Meng , 6 Qi-Qi , pro- ) 2 , Posted on Authorea 2 Aug 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.162790498.82247747/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. ytm n erdciepten.Gncoimadhrahoiimaetems motn reproductive important determination most sex the of are variety a hermaphroditism exhibit and fishes Gonochorism environment, water patterns. the reproductive in and niches systems ecological of diversity the to Due Introduction evolution hermaphrodite for Words: information valuable Key provides also but evolution aquaculture. hermaphrodite fish of enhancing underpinnings of genetic evolution the sequence into the insight by explained teleosts be other may in lost was or a analysis. Larimichthys revealed transcriptome the which in analysis, genomic Phylogenomic The comparative fusion polyactis or species. genes. enabled fission two chromosome protein-coding the assembly major between 25,233 without events genome translocations and and high-quality rearrangements Mb) within-chromosome of 4.52 Our number = N50 ago. scaffold years and Mb that suggests 1.21 analysis = of N50 relationship evolutionary (contig and Mb origin the Asia, East crocea in ( Larimichthys species croaker fish yellow commercial little famous the most makes stage polyactis hermaphroditic L. transient transcriptome, special The gonadal solved. sequence, whole-genome Abstract: croaker, yellow evolution hermaphrodite little Keywords: 8 number: Figure croaker yellow count:7701 Word little of research Genome title: China; Running 310021, Lou) Hangzhou (B. Sciences, [email protected] Agricultural of addresses: Academy E-mail Zhejiang Hydrobiology, of Institute Lou: *Bao Alabama Biotechnology, for University; Institute addresses: Auburn Alpha E-mail Hudson Medicine, The Veterinary USA; USA; 35806, of 36849, Alabama AL College Huntsville, Auburn, Pathobiology, Station; of Experiment Agricultural Department Sciences, Wang: Earth *Xu and Ocean of College addresses: Science, E-mail Environmental China; Marine 361005, of Xiamen Laboratory University, Key Xiamen State Xu: *Peng authors: Corresponding author. Corresponding * Xiamen China; Sciences, # 100016, Earth Beijing and Institute, Ocean Bioinformatics of Novogene College 6. Science, Environmental Marine China; 361005, of Xiamen Laboratory University, Key State 5. hs uhr r otiue qal oti work this to equally Contributed are authors These h xrsinof expression The . h vltoaydrcino oohrs n emprdts sa nrgigmseyt be to mystery intriguing an is hermaphroditism and gonochorism of direction evolutionary The napaigmdlfrsuyn h omto fhrahoie.O h te ad sthe as hand, other the On hermaphrodites. of formation the studying for model appealing an ) u o nohrtlotseis ugsigta toiiae rmalnaeseicduplication lineage-specific a from originated it that suggesting species, teleost other in not but iteylo rae,woegnm eune oaa transcriptome, gonadal sequence, whole-genome croaker, yellow little [email protected] [email protected] eanucer ee erpr h eoesqec of sequence genome the report we Here, unclear. remain .polyactis L. . hlgntcaayi hw httehrahoiesaei male in stage hermaphrodite the that shows analysis Phylogenetic dmrt1 Rnf183 iegdfo h omnacso of ancestor common the from diverged n t ptemrgltr gene regulatory upstream its and dmrt1 X Wang) (X. P Xu) (P. nieistoparalogues two its unlike , eewsietfida h addt e eemnto eein gene determination sex candidate the as identified was gene dmrt1 2 eoigthe Decoding . rnf223 .polyactis L. rnf183 aiihhscrocea Larimichthys .polyactis L. and eebt eulydimorphic sexually both were rnf225 eoentol provides only not genome aiihhspolyactis Larimichthys sol rsn in present only is , ihasz f˜706 of size a with , dmrt1 dmrt1 .polyactis L. 2. million ˜25.4 .polyactis L. , , rnf183 rnf183 and L. , , , Posted on Authorea 2 Aug 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.162790498.82247747/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. eess n h icvr fatasetjvnl nesxsaei ae loerce h hoyo teleost of theory the have enriches We also males sexes. in in both stage gonochorism intersex from and juvenile points transient hermaphroditism time a between development of relationship discovery gonadal the the 18 studying and for and teleosts, model organs excellent major hermaphrodite of an in patterns 13 Mb) established architecture of reproductive (706 genomic from genome its genome of obtained assembled in role reference the evolution high-quality high-quality also elucidate a and A to structure produced study, that 2019). we this genome revealed In evolution, Gil, of analysis and studies methods. karyotype differentiation Park for sex and 2020; toolkit and necessary map al., the linkage et provide established (Liu genetic will successfully 48) a was = of programme (2n report diploid breeding previous artificial A an wild in 2015, 2021). of miniaturization In spawning growing artificial resources. and by fishery imbalance, restore gender precocity, to sexual needed changes of caused quality resources has the water overfishing Although polyactis and 2004), 2010). current al. ocean al. et and et Han pollution, 2016; seawater al., et overfishing, level. 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Ntoa etrfrBoehooyIfrain[CI Taxonomy [NCBI] Information Biotechnology for Center (National eoesz ae nte1-e rqec itiuinuigthe using distribution frequency 17-mer the on based size genome eue ed rmpie-n irre odtriethe determine to libraries paired-end from reads used we , .polyactis L. 4 oa f˜3 bo eunigdt was data sequencing of Gb ˜431 of total A . https://www.ncbi.nlm.nih.gov × L eoislne ed,wr also were reads, linked Genomics . polyactis eoefiehsbeen has file genome ,adthe and ), Posted on Authorea 2 Aug 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.162790498.82247747/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. eemdl eeae rmEiecMdlrwt h rncit sebe yTiiy(rbere al., the et adjusting (Grabherr after Trinity structures by gene annotation structures assembled final Functional gene transcripts the all the acquire nonredundant to with merged the 2003) EVidenceModeler We generate al., 2011). to from and software. et 2008) 2011) generated 2003) (Hass al., al., models PASA al., et et used gene (Haas et we (Grabherr EVidenceModeler Finally, (Hass 2.1.1) using sets. PASA (version approaches gene Trinity by three by the structures data positions. by alignment gene RNA-seq obtained on the the based assembled 2010) we obtained al., we hand, then et one hand, (Trapnell the other 2.1.1) On (version the prediction. cufflinks On gene by structures in the gene assist obtained to to and used data were 3.0.2) RNA-seq tissues the (version different 1.0) mapped from GlimmerHMM (version data methods. 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No reuse without permission. — https://doi.org/10.22541/au.162790498.82247747/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. musculus hlgntcrltosisa h eoelvlwr nlsdfregte species, , eighteen for analysed , were level genome found 2016). , the we al., levels at Then, et genome relationships acids. 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All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.162790498.82247747/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. )(LSP RRID:SCR (BLASTP, 8) m crocea to Similar the of Comparison the in crocea polyactis aculeatus and analyses. Gasterosteus selection branch the positive foreground were in of the found P-values groups were as three genes 2013). ran selected We al., positively set 955 we et selection. group, positive Zhang first undergone the 2005; have In to al., thought et were different (Zhang 0.05 calculated model We the branch-site using the computed 2012). 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Homo and , and , , L. L. L. Posted on Authorea 2 Aug 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.162790498.82247747/v1 — This a preprint and has not been peer reviewed. 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(Figure branch and eeietfidi h e eemnto ein(iue4) .2M einsurrounding region Mb 4.62 A 4A). (Figure region determination sex the in identified were , to L L .polyactis L. rnf122 h eut hwdthat showed results The . . . crocea polyactis in ) eaae rmteacso of ancestor the from separated .crocea L. .polyactis L. Fgr B.Snealrenme of number large a Since 4B). (Figure eoeasml.Atog hs hoooe i o xeinechromosome experience not did chromosomes these Although assembly. genome L . polyactis , atrsesaculeatus Gasterosteus .polyactis L. a oprdt h orsodn ytncrgo in region syntenic corresponding the to compared was rnf and .crocea L. .crocea L. aiymmeshv ihdge fhmlg ewe these between homology of degree high a have members family L .polyactis L. . ee nlddi h otatdo xaddfmle were families expanded or contracted the in included genes crocea lnaegop2,L2)and LG22) 22, group (linkage 9 L ieg 2. ilo er g.Teacso of ancestor The ago. years million ˜25.4 lineage .fugu T. . eeldta 4crmsmsof chromosomes 24 that revealed crocea , ai rerio, Danio dpain Fgr ,SplmnayTables Supplementary 2, (Figure adaptations prxmtl 01mlinyasao Gene- ago. years million 90.1 approximately Fgr ) h eut eetdtehigh the reflected results The 3). (Figure rnf ee eentosre nayother any in observed not were genes rnf and .polyactis L. ee n uooa members autosomal and genes .polyactis L. .polyactis L. rnf .polyactis L. aiyadseveral and family ) n htteother the that and L .polyactis L. dnie core a identified . .crocea L. polyactis cfap157 (chromosome lineage dmrt1 could was , (4.73 and L L. . Posted on Authorea 2 Aug 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.162790498.82247747/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. n ihia,sgetn eels uigeouin(iue7A). (Figure evolution during loss gene suggesting Cichlidae, and Although amphibians. and examined, reptiles, mammals, of in history evolutionary the investigated genes, We effect. damage collateral to compared Rnf183 its in profiles transcription the Rnf183 of analysis Further from of 6C). gene, away transcripts (Figure upstream the of insertion immediate Analysis 18-bp an dimorphic. an identified has neighbourhood exon gene fourth the , that , of indicated structure results gene The The 6B). (Figure region this in in evolution genomic rapid sting related closely an the with Between rearranged, was order of gene orientation gene the that showed smarca2 map the locus in gene shown A are reptiles, species that mammals, these includes indicated of group hermaphroditism. results patterns main and These reproductive other The 6A). The gonochorists. (Figure branch. are diagram separate which a amphibians, in and distributed branches. birds, is different and in group scattered are aphrodite they and gonochorists, typical calcarifer Lates of merra branches Epinephelus the Interestingly, protogyny. crocea hermaphrodite except Perciformes, and are protandry them hermaphrodite of Most 6A). (Figure group polyactis main one in clustered for be could analysed from species constructed fish was tree phylogenetic A that conclude and we 6), (Figure Therefore, stages biased. all in testis-specific sex was of panel A and dph). clustered (50-720 them, dph) together Among (240-360 clustered maturity development sexual ovarian including of and of genes, testis stages differentiation-related functional and clustered all clustered the dph) determination- dph dph, 720 form 40 and 50 to before 400 40 From (50-180, meiosis gonads at meiosis together. early-stage of gonads before The testes initiation sex Methods). and indistinguishable and dph (see dph, 40 together; stages 30 at later and gonads 13 sex 20, indistinguishable at 10, ovaries together; conducted 7, also and at was testes samples profiling and whole-fish expression dph, gene in Gonad stages intestine. developmental and ovary, 18 testis, at skin, heart, eye, brain, thirteen gene spleen, on candidate performed sex-determining was analysis the Transcriptome of analysis expression and Identification species, two .latipe O. Larimichthys rza latipes Oryzias dmrt1 dmrt1 eelctdbtenseiswt emprdt rtnr n emprdt rtgn,including protogyny, hermaphrodite and protandry hermaphrodite with species between located were rnf223 s3k ptemof upstream kb 3 is n t ls aaousso atr frpdeouini teleosts in evolution rapid of pattern a show paralogues close its and scnevdaogmmas etls mhbas n te eess except teleosts, other and amphibians, reptiles, mammals, among conserved is ae on based eeanttdi h eoeadtasrpoedt Fgr n upeetr iueS1). Figure Supplementary and 5 (Figure data transcriptome and genome the in annotated were , rnf183 dmrt1 hwdasgicn etsba.I ersrce h ee ntesxdtriainregion, determination sex the in genes the restricted we If bias. testis significant a showed , .rubripes T. .polyactis L. dmrt1 y1aa ga rnf183, figla, cyp19a1a, and n o nayohrtlotspecies. teleost other any in not and and a nyb on nSieia,Ltde oaetia,Lbia,Saia,Salmonidae, Sparidae, Labridae, Pomacentridae, Latidae, Sciaenidae, in found be only can Fgr 6D). (Figure , rnf225, dmrt1 , t vr-pcfi xrsinmyrsl ntsi-pcfi xrsinof expression testis-specific in result may expression ovary-specific Its . ootrsalbus Monopterus rpoeismarmoratus, Kryptolebias pnpeu coioide Epinephelus dmrt1 , and .niloticus O. sstill is r lsl eae aaous rhlge o l he ee ol efound be could genes three all for Orthologues paralogues. related closely are .rupries T. dmrt1 .crocea L. otis5eos eebigta fohrtlotseis including species, teleost other of that resembling exons, 5 contains .crocea L. rnf183 n tdslyda poiesxbae atr ngndexpression gonad in pattern sex-biased opposite an displayed it and , dmrt1 and . and Fgr 4C). (Figure and rnf183 rohoi niloticus Oreochromis , dmrt1 arsbergylta Labrus Larimichthys eeisrinimdaeyusra of upstream immediately insertion gene stebs e eemnto eecandidate. gene determination sex best the is foxl2 h s ru otisgncoit ueiehermaphrodite, juvenile gonochorist, contains group fish The . .crocea L. rnf183 and eeldta hsgn a w xn n sol 17bases 3197 only is and exons two has gene this that revealed uloiesqecs h eut hwdta oto the of most that showed results The sequences. nucleotide hwda vr xrsinba,while bias, expression ovary an showed .polyactis L. dmrt1 rnf223 ootrsalbus Monopterus 10 Fgr )and 7) (Figure dmrt1 oprdwt hto t ls relative close its of that with Compared . h ptemgn otnswr ieet sugge- different, were contents gene upstream the , a orltdwt h vlto fgonochorism of evolution the with correlated was y1aa ga n13 ol,rf2,gd,amh, gsdf, rnf225, foxl2, rnf183, figla, cyp19a1a, , and etorsi striata Centropristis n t ontemgenes downstream its and ise,gl,msl,kde,ha-iny liver, head-kidney, kidney, muscle, gill, tissues, rnf183 , rnf225 rza latipes Oryzias ai rerio Danio figla rnf183 hs xrsinwsas sexually also was expression whose , iial,tecoetrltv of relative closest the Similarly, . r rsn nms s pce we species fish most in present are Fgr )wr infiatyovary significantly were 8) (Figure n on httoautosomal two that found and eog otejvnl herm- juvenile the to belongs and , cnhpgu schlegeli Acanthopagrus dmrt1 rnf183 aiuurubripes Takifugu dmrt3 .polyactis L. 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Data may be preliminary. n t aaouscnb dnie n noae,wihpoie nte niaino h ihqaiyof quality high Genomics the 10x of level. and indication pseudochromosome Bionano another the provides using to which mapping annotated, assembly Physical and genome 2019). identified assembly. the al., be gene of can 2019; et using al., quality paralogues Yang species et its the 2019; Ge fish and 2019; improved al., several al., further of et et Conte assembly reads Jiang 2019; genome linked al., 2019; contig the et al., The than Cai assembly. 2018; contigs et al., genome of He et number the (Gong smaller S17) of (Table a sequencing completeness with third-generation and and longer assembly accuracy much long-read is the of N50 combination ensured The correction technologies. high-quality short-read sequencing first Illumina long-read the from of is benefited assembly genome This of Our technologies. contiguous studies Genomics most biological 10x the for and assembled crucial Bionano, novo we is sequencing, study, genome long-read present reference nanopore the A In males. than . larger remarkably females polyactis L. of assembly genome high-quality A in dph) (43-80 Discussion stage transient mechanism. the hermaphrodite molecular in transient and testis the basis the formation that than in ovary indicated expressed testis. of the results all in levels the were These expressed genes expression in highly three than the more these ovary was in Interestingly, in it dph) the stages. significantly (43-80 whereas few in was stage gonad, a level hermaphrodite expression and at higher brain, its testis significantly eye, cases, the gill, a in some the maintained in in and dph, expressed dph mainly 7 40 as at testes. early in detected as than expressed ovaries and in Although higher differentiation. tissues ovarian breeding many and the in formation, entering oocyte expressed when synthesis, stages ovary oestrogen later the to and in related dph) detected (7-40 also to juvenile was the contrast figla gsdf dph). during In 720 of expression and expression testis. high (360 high season maintained mature However, and dph). to dph 50 spermatogenesis 7 (after as during early downregulated as was expressed it First, profiles. and testis. expression dph), the analyse in to expressed highly screened and were genes sex-related studied (anti-M and well-identified few A in profiles expression gene Sex-related rnf183 While duplication. of divergence (RPKM functional testis tissues. the other in and level skin in detected sion (RPKM of expression gonadal analysed, and distribution tissue with The species fish other of that teleosts. from among of not location but sex-linked of reptiles orientation and gene amphibians, rnf223 the mammals, that among showed conserved map relatively locus gene A fco ngrln lh,o h.2 and 2) Chr. on alpha, germline in (factor sebe eoei candeuigtidgnrto sequencing. third-generation using Sciaenidae in genome assembled pcaie nsxdtriain ieetaino aneac nteoayof ovary the in maintenance or differentiation determination, sex in specializes leinhroe nCr )and 6) Chr. on hormone, ullerian in ¨ > rnf183 Xenopus 120). sacmecal motn candefihseis dl s hwsxa iopim with dimorphism, sexual show fish Adult species. fish Sciaenidae important commercially a is rnf225 a xrse tlwlvl nglstse n a xrml ihyepesdi ovaries in expressed highly extremely was and gills/testes in levels low at expressed was rnf223 ugsigta hymyhv endrvdfo nacetdpiaineet The event. duplication ancient an from derived been have may they that suggesting , rnf183 a ihyepesdi il (RPKM gills in expressed highly was rnf183 Cyp19a1a and cyp19a1a > sol on in found only is 0 u oepeso nteoay(iue7) hs eut niae the indicated results These 7C). (Figure ovary the in expression no but 10) rnf225 Figla , Amh rnf223, rnf183 .polyactis L. and ctcrm 40 aiy1,sbaiyA oyetd a nCr 10), Chr. on 1a, polypeptide A, subfamily 19, family P450, (cytochrome smil xrse ntegnd,epcal nteoay twsfirst was It ovary. the in especially gonads, the in expressed mainly is ananterepeso n ucini h iladohrtissues, other and gill the in function and expression their maintain ntae t xrsinfo 0dhbfr etclrmiss(50-180 meiosis testicular before dph 40 from expression its initiated rnf223 L foxl2 gsdf and rhlge Fgr 7B). (Figure orthologues . polyactis .polyactis L. Larimichthys gndlsm-eie atr nCr 6 r gonad-specific are 16) Chr. on factor, soma-derived (gonadal rnf225 ntetsi n vr,except ovary, and testis the in foxl2 a xrse ral nmlil ise,wt moderate a with tissues, multiple in broadly expressed was n vna hssae hr een infiatdifferences significant no were there stage, this at even and , rnf183 11 Frha o 2 nCr 5 r osdrdtightly considered are 15) Chr. in L2, Box (Forkhead rsmbydet ufntoaiainatrgene after subfunctionalization to due presumably , rnf183 , rnf223 n t eenihorodi eydifferent very is neighbourhood gene its and , > n t ptemaddwsra ee is genes downstream and upstream its and 2) ihol eylwlvlo expres- of level low very a only with 120), o h.1) and 11), Chr. (on .polyactis L. foxl2 .polyactis L. rnf225 i h nerto of integration the via t5 p Fgr 8). (Figure dph 50 at cyp19a1a .polyactis L. rnf183 m,Gsdf amh, Cr 4 were 24) (Chr. a complex a has .polyactis L. a widely was Foxl2 snx to next is . rnf183 Amh was was de Posted on Authorea 2 Aug 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.162790498.82247747/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. o tdigteeouinr ieto ewe emprdts n oohrs.Wehrtesexual the Whether gonochorism. and hermaphroditism between direction evolutionary the studying for except , and the 2004; of availability Cheng The and Lin 1996; hermaphroditism. (Jin to in gonochorism decades Therefore, pattern from recent 2020). in differentiation al., grounds et gonadal spawning Lu in this disruptors that endocrine environmental above, speculate mentioned We As demonstrated ancestor. very 2021). study gonochoristic previous is al., Our Perciformes spectrum. et in reproductive gonochorism fish (Xie the and of hermaphroditism evolution from the of transition that of evolution the study the the for in for reasons important species be transitional may a These Finding disruptors population. endocrine the environmental throughout of hermaphroditism. effects ratio females to the sex as resist gonochorism balanced such to imba- maturation, a help may sexual maintain ratio or accelerated to sex males, to population-level contribute before from may meiosis a pollution genes initiating environmental in always sex-related alternatively, resulting some earlier; individuals in feminization, mature larger Mutations to cause lance. pressure, forced changes. may fish are fishing environmental disruptors and teleost to perhaps endocrine capture that adapting environmental changes, escape suggesting of to environmental likely species, process various less the from hermaphroditic are in stress of across independently widespread experiencing solely reproduction is After consists sexual hermaphroditism order own literature, evolution no their the the evolved study and in to phylogeny, reported models fish excellent As as teleost serve gonochorism. could and fish reproduc- hermaphroditism these of simultaneous and of types 2009), and all protogyny, Mank, have and hermaphrodite lack Perciformes protandry, (Avise the hermaphrodism methods. hermaphrodite to reproductive gonochorism, due two including unclear above systems, remains the gonochorism tive between and hermaphroditism species plants between transitional in to evolution of times of due many direction re-evolved gonochorism the and fish, from lost In evolve been 2010). have could SS, loci hermaphroditism Renner or exists; genes H, hermaphroditism determination also (Schaefer mutations from sex female-sterility opposite and evolve or a The reproduction, male- could of sexual 2014). on occurrence gonochorism al., the endpoints where or et investment evolutionary (Bachtrog 2009), sex-specific two increasing Mank, gradually the by and fish either are (Avise in hermaphroditism gonochorism spectrum simultaneous and reproductive and hermaphroditism of gonochorism comparisons. evolution Theoretically, pairwise and the in fission studying found chromosome for were experience translocations model not and did A rearrangements chromosomes of these number although a species, events, of close fusion genome these the of in genomes assembled the contain chromosomes the genomes of with haploid number consistent their The was and chromosomes. (TGD), 25 duplication to genome 24 teleost-specific undergone have teleosts Most con- of genome genomes the addition, the aculeatus In than identified. lower the was also were which in lanceolatus RNAs sequences, annotated non-coding repetitive were 5,671 22.28% of tained genes total protein-coding A annotations. 25,233 functional of total A w lsl eae pce hwdta lotalcrmsmsof chromosomes all of LG22 almost chromosome that sex showed The species homology. related closely two while stage, hermaphrodite processes. transient a development gonad and whereas maturity, sexual patterns reproductive in differences .crocea L. .polyactis L. .polyactis L. (25.2%). 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Data may be preliminary. two,T . ek .E 19) RNSapoenmtffigrrn database. fingerprint informati- motif protein sequence PRINTS–a Amini, long-range (1994). M., E. vitro, M. Ronaghi, In Beck, 7 & L., (2014). K., Christiansen, T. J. contiguity. Attwood, A., transposase Shendure, Kumar, via & assembly R., J., genome Daza, F. novo ps://doi.org/10.1101/gr.178319.114 N., de Steemers, J. for L., on Burton, K. programs. O., Gunderson, search J. S., database Kitzman, protein A., of Adey, generation new a PSI-BLAST: research Sch and L., BLAST T. Gapped Madden, F., S. life. Altschul, daily co. seedling and aquatic materials harbor experiment Xiangshan of the convenience References from for Veterinary Xu of China Wantu College Mr. Ningbo, University County, and Auburn Xiangshan from Zheng LTD, fund Genxing start-up Mr. laboratory thank generous Station We a Experiment Medicine. Foundation Agricultural as Science Alabama well National an as 1018100), 1928770), Grant, by OIA Enabling project (NSF supported (Hatch Fellowship Agriculture is Research technology Track-4 and X.W. (grant RII and Food 2019C0001). EPSCoR of China the number science Institute (grant LQ19C190002); of granary National county number USDA Xiangshan (grant Foundation blue the Province of of Science Department Zhejiang Technology of project Natural and Foundation special Science Science National Natural and the the national 31902341); 2018YFD0901204); key number number the by (grant innovation supported was work This dimorphism expression gene sex-related Acknowledgments for foundation solid a provides also in but evolution hermaphrodite of of genome high-quality that the summary, demonstrated In results These dph. 130 before stage. samples testis and ovary figla between difference significant no the whereas however, dph, 80 stage; after hermaphroditic Interestingly, transient testis. the male-specific respectively in tilapia, levels XX lower in male 2017). to al., In female et play from Zhang Dmrt1 2016, reversion al., and sexual Foxl2 et and bind 2016). (Lau as zebrafish primarily al., early in (BPA) as et masculinization A expressed (Xie complete bisphenol is ovary or Cyp19a1a the E2 tilapia, of of action In oestrogen differentiation 2019). zebrafish, the al., maintaining In regulating drive et et by 2013). and to Wang differentiation Li development sex fish 2013; 2018; in ovarian female al., Chang, roles antagonistic promoting et XX and Li in (Wu in 2010; dph teleosts role differentiation al., 5 gonochoristic vital sex et and Guiguen in a hermaphroditic 2007; that play factor al., both documented key 1) in well a Member afterward is considered A feminization It is Subfamily 2000). oestrogen 19 Nagahama, al., differentiation, Family 2007; et ovarian al., (Qin of mutant et inducer the (Wang follicular in natural individual nucleolar phenotype a to chromatin all-male As oocytes an prefollicular CN in the cystic resulting at blocked, from was transition oocytes 2018). IB) subsequent forming (stage the oocytes However, meiosis, perinucleolar IA). underwent (stage and stage cysts (CN) 2008). in al., clustered et were (Jørgensen zebrafish, occurred In differentiation 2015). gonadal al., et (Qiu spermatids 7,8188 ( 841–848. (7), .polyactis L. .polyactis L. , foxl2 . dmrt1 25 and , 1) 3930.( 3389–3402. (17), hsi aubeifrainfrehnigfihaquaculture. fish enhancing for information valuable is This . , Sn ta. 00.Ls ffnto fcp91 yCIP/a9adTLN illa to lead will TALENs and CRISPR/Cas9 by cyp19a1a of function of Loss 2020). al., et (Song https://doi.org/10.1093/protein/7.7.841 cyp19a1a figla foxl2 , foxl2 r epnil o P omto n h aeseictasethermaphroditic transient male-specific the and formation EPO for responsible are and and cyp19a1a https://doi.org/10.1093/nar/25.17.3389 cyp19a1a ffr .A,Zag . hn,Z,Mle,W,&Lpa,D .(1997). J. D. Lipman, & W., Miller, Z., Zhang, J., Zhang, A., A. ¨ affer, ). .polyactis L. figla xrsinee ece eyhg ee t10dh u hr was there but dph, 100 at level high very a reached even expression figla eeas bnatyepesdi h vr n xrse at expressed and ovary the in expressed abundantly also were , foxl2 a xrse rm2 p,wihwslkl h iewhen time the likely was which dpf, 26 from expressed was cyp19a1a o nypoie nih notegntcunderpinnings genetic the into insight provides only not 14 and cyp19a1a ). figla figla xrsinadosrgnpouto L tal., et (Li production oestrogen and expression eoeresearch Genome uatzbas hwdta h emcells germ the that showed zebrafish mutant ee erae ihoct degeneration oocyte with decreased level eeas xrse ntetsi nthe in testis the in expressed also were foxl2 Esr2a ). and cyp19a1a oihbtteepeso and expression the inhibit to , 24 1) 0124.( 2041–2049. (12), rti engineering Protein Ctcrm P450 (Cytochrome uli acids Nucleic htt- . Posted on Authorea 2 Aug 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.162790498.82247747/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. da .C 20) UCE utpesqec lgmn ihhg cuayadhg throughput. high and accuracy high with mammalian alignment research of sequence evolution multiple acids The MUSCLE: (2006). (2004). W. C. M. R. Edgar Hahn, & N., Cristianini, E., J. families. Stajich, gene T., teleosts. Bie, De in P., J. hermaphroditism Demuth, Functional (2008). M. Liu, ( of 1-43. study and the Y.S. for tool Mitcheson, computational a De heuristics CAFE: (2006). new W. models, M. Hahn, more evolution. & P., family 2: J. gene Demuth, jModelTest N., (2012). Cristianini, T., D. Bie, De Posada, from & assemblies R., computing. Doallo, genome parallel L., microbial and G. finished Taboada, Nonhybrid, D., (2013). Darriba, al. et data. 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English with Chinese (In ) . uli cd research acids Nucleic 10 6–6.( 563–569. , https://doi.org/10.1093/nar/28.1.45 15 ). https://doi.org/10.1038/nmeth.2109 ). suocan polyactis Pseudosciaena https://doi.org/10.1093/bioinformatics/btl097 LSBiol. PLoS en/CJFDTOTAL-ZSCK200406012.htm https://doi.org/10.1038/nmeth.2474 auegenetics Nature 12 ). . eoeresearch Genome 33 7:1089 ( (7):e1001899. Dtbs su) D212–D215. issue), (Database uli cd research acids Nucleic ). ihadFisheries and Fish . bioRxiv e Dev Sex ihSci. Fish . 25 o:10.1371/jour- doi: ora fFishery of Journal ). . ). 1,2–9 ( 25–29. (1), 14 2004.934711. , . 5,988–995. (5), 35 3 (2-3):152- :250-254. Nucleic ). (In ) , , htt- 9 27 ). : Posted on Authorea 2 Aug 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.162790498.82247747/v1 — This a preprint and has not been peer reviewed. 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(1), 16 < http://www.repeatmasker.org/RepeatModeler.html erdciebooyadedciooy:RB&E : endocrinology and biology Reproductive https://doi.org/10.1186/gb-2008-9-1-r7 e opEndocrinol Comp Gen ) auebiotechnology Nature uli cd research acids Nucleic https://doi.org/10.1093/nar/gkt1223 . . 34 33 Dtbs su) D227–D230. issue), (Database Dtbs su) D121–D124. issue), (Database . 32 4,8585 ( 835–845. (4), ). . 6:5–6 (doi: 165:352–66. 29 . 31 7,644–652. (7), 1) 5654– (19), ). htt- ). , Posted on Authorea 2 Aug 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.162790498.82247747/v1 — This a preprint and has not been peer reviewed. 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Q.X. analyses: legends paper: data F.L.; version. the Figure the W.Z., submitted wrote H, performed the X.W; X. approved S.; and and Q.X., J. X. read P. sample: and revision, L., collected H. Q. B.L.; X. L., and Q.X., M. X.W. experiments: Q.X., the experiments: performed the designed and Conceived interests. competing Contributions no Author have version they is that declare paper authors this The in described Interest (SRA) of version Archive Conflict The Information, Read of Biotechnology Sequence JAFMOB000000000. assembly for the number chromosome Center JAFMOB000000000. in accession final (National deposited under The NCBI been Assembly to have PRJNA705840. Female submitted data number in been sequencing Results Bioproject cyp19a1a RNA under or and foxl2 database genome of Mutation raw (2017). The al. et M, Tilapia. 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H., a 10(6), Wang, Is R., RNF183 Feng, ligase K., Ubiquitin Chao, S., Zhang, Q., Yu, https://doi.org/10.1093/molbev/msi237 itr hwn eaeadml iteylo croaker, yellow little male and female showing picture A (Lcr), (Gac), (Tru), aiihhspolyactis Larimichthys rohoi niloticus Oreochromis (Gga), ai rerio Danio yolsu semilaevis Cynoglossus (Gmo), https://doi.org/10.1093/ecco-jcc/jjw02 Science rnhotm floridae Branchiostoma in intestinalis Ciona De and (Dre) . 339 ). o clResour. Ecol Mol 61) 5–6.( 456–460. (6118), aiihhspolyactis Larimichthys aiihhspolyactis Larimichthys (Oni), (Cse), ). Endocrinology. (Cin), atrsesaculeatus Gasterosteus ai rerio Danio (Bfl), enovo de nlscarolinensis Anolis 16 eiotu oculatus Lepisosteus 22 oeua ilg n evolution and biology Molecular https://doi.org/10.1126/science.1230835 5-6.( 755-768. oooybsd N-e-ae) vr8.%of 84.3% Over RNA-seq-based). homology-based, , (D) ) (Dre), 5()23–7 di 10.1210/en.2017-00127). (doi: 158(8):2634–47. (Lpo). h endarmsoscmo n unique and common shows diagram Venn The polyactis L oosapiens Homo aiihhscrocea Larimichthys aiihhspolyactis Larimichthys eoeassembly. genome https://doi.org/10.1111/1755-0998.12476 (C) (E) (Gac), Aa and (Aca) rti rhlg oprsnamong comparison orthology Protein iegnedsrbto fclassified of distribution Divergence . (Loc), (B) rza latipes Oryzias https://www.ncbi.nlm.nih.gov (Hsa), oprsno h eesets gene the of Comparison alricu milii Callorhinchus eou tropicalis Xenopus (Lcr), u musculus Mus . 22 (Lpo), 1) 2472–2479. (12), atrsesac- Gasterosteus .polyactis L. (Ola), ). Larimichthys Takifugu (Mmu), (Cmi), (Xtr). has ). ) Posted on Authorea 2 Aug 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.162790498.82247747/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. 3,1020 7,30 6,40ad70dh(asps acig eesqecduigIlmn 00HiSeq 2000 100, Illumina 80, using sequenced 70, were 60, hatching) 50, post 40, (days 30, dph 20, 720 10, and 7, 400 from 360, testes 340, and 270, ovaries 240, of 180 pairs 130, thirteen and line stages black sex of the indistinguishable profiles below from away expression numbers bp The and 3197 distribution 5. the is exon 3, and represents exon exons represents rectangle bases. two rectangle red of green the number dark and the the 4 2, represent exon exon represents represents browser. rectangle rectangle green the blue light to the according 1, described exon annotation. represents are genome symbols the on Gene based figure. are this make to used ( croaker yellow little ( musculus indicate circles red and ago, years of millions in num- fossil The times from diverge genes. carolinensis estimated time orthologous the single-copy calibration indicate 592 the branches using constructed the genomes on vertebrate of bers 18 analysis of contraction tree and Phylogenetic expansion gene and Phylogenetic 2. 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Tissue Anolis and ) chro- Mus has rnf . Posted on Authorea 2 Aug 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.162790498.82247747/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. al umr of Summary 2 Table for data sequencing of statistics P The at 1 difference Table significant a represent ** and Tables * test. P hoc at indicate respectively, P of post bars points, and sample error Duncan’s time 0.05 a the by different from above followed at SD ANOVA letters +- males one-way uppercase means and by and the lowercase females as Different in presented differences point. are significant data results each The for testis. individuals and three ovary the of stages developmental of significant in profiles a profile Expression represent expression ** gene Sex-related and * 8. Figure test. 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(B) rnf183 isedsrbto n xrsinpolsof profiles expression and distribution Tissue , ytncaaye of analyses Syntenic < gsdf < nlscarolinensis Anolis .1btenfmlsadmls epciey yidpnett-test. independent by respectively, males, and females between 0.01 , .1btenfmlsadmlsa h aetm on,rsetvl,by respectively, point, time same the at males and females between 0.01 ae calcarifer Lates rnf223 , cyp19a1a eoeasml statistics assembly genome , n rnf225 and dmrt1 , gaadfoxl2 and figla rnf183 ,adlre( large and ), ieetupraeadlwraeltesaoeteerror the above letters lowercase and uppercase Different . ,fos( frogs ), .polyactis L. 24 nvrerts h vltoayhsoywsinferred was history evolutionary The vertebrates. in .polyactis L. n t daetgnsi uas( humans in genes adjacent its and eou laevis Xenopus aiihhscrocea Larimichthys ae ntsu itiuin n different and distributions tissue on based .polyactis L. eoeAssembly genome rnf183 gonads. rnf183 ,tlpa( tilapia ), n t paralogues its and eesmosaebsdon based are symbols gene ’ , rnf223 n iteylo croaker yellow little and ) rohoi niloticus Oreochromis < .5 sdetermined as 0.05, and oosapiens Homo rnf225 < < rnf223 n in and 0.05, 0.05, < ), ), Posted on Authorea 2 Aug 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.162790498.82247747/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. iue2. Figure 1. Figure Figures Guanine/Cytosine GC Ccnet41.49% bp 625,267,019 bp 102% 706,148,513 5,671 25,233 21.26% 22.28% 309 88.55% characteristics Genome chromosomes on chromosomes anchored to scaffolds anchored of is Length genome the (0.575%) chromosomes of 3,895,963 on Proportion anchored scaffolds of number Quantity gene RNA non-coding Predicted kb) number per gene (SNPs protein-coding rate Predicted and number SNP Heterozygous elements transposable of Content elements repetitive of Contents content GC assembled Percent characteristics Genome length Total 25 Posted on Authorea 2 Aug 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.162790498.82247747/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. iue3. Figure 26 Posted on Authorea 2 Aug 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.162790498.82247747/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. iue4. Figure 27 Posted on Authorea 2 Aug 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.162790498.82247747/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. iue5. Figure 28 Posted on Authorea 2 Aug 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.162790498.82247747/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. iue6. Figure 29 Posted on Authorea 2 Aug 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.162790498.82247747/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. iue8. Figure 7. Figure 30 Posted on Authorea 2 Aug 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.162790498.82247747/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. 31