An Example from the Floral Epidermis Ofthe Nymphaeaceae

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Year: 2018

Disentangling historical signal and pollinator selection on the micromorphology of flowers: an example from the floral epidermis ofthe Nymphaeaceae

Coiro, Mario ; Barone Lumaga, Maria Rosaria

Abstract: The family Nymphaeaceae includes most of the diversity among the ANA‐grade angiosperms. Among the species of this family, floral structures and pollination strategies are quite varied. The genus Victoria, as well as subgenera Lotos and Hydrocallis in Nymphaea, presents night‐blooming, scented flowers pollinated by scarab beetles. Such similar pollination strategies have led to macromorphological similarities among the flowers of these species, which could be interpreted as homologies or convergences based on different phylogenetic hypotheses about the relationships of these groups. We employed SEM of floral epidermis for seven species of the Nymphaeaceae with contrasting pollination biology to identify the main characters of the floral organs and the potential homologous nature of the structures involved in pollinator attraction. Moreover, we used TEM to observe ultrastructure of papillate‐conical epidermis in the stamen of Victoria cruziana. We then tested the phylogenetic or ecological distribution of these traits using both consensus network approaches and ancestral state reconstruction on fixed phylogenies. Our results show that the night‐blooming flowers present different specializations in their epidermis, with Victoria cruziana presenting the most elaborate floral anatomy. We also identify for the first timethe presence of conical‐papillate cells in the order Nymphaeales. The epidermal characters tend to reflect phylogenetic relationships more than convergence due to pollinator selection. These results point to an independent and parallel evolution of scarab pollination in Nymphaeaceae, and show the promise of floral anatomy as a phylogenetic marker. Moreover, they indicate a degree of sophistication in the anatomical basis of cantharophilous flowers in the Nymphaeales that diverges from the most simplistic views offloral evolution in the angiosperms. This article is protected by copyright. All rights reserved.

DOI: https://doi.org/10.1111/plb.12850

Posted at the Zurich Open Repository and Archive, University of Zurich ZORA URL: https://doi.org/10.5167/uzh-151916 Journal Article Accepted Version

Originally published at: Coiro, Mario; Barone Lumaga, Maria Rosaria (2018). Disentangling historical signal and pollinator selection on the micromorphology of flowers: an example from the floral epidermis of the Nymphaeaceae. Plant Biology, 20(5):902-915. DOI: https://doi.org/10.1111/plb.12850 Italy80139 Napoli, 2 [email protected] 107, 8008Zurich,Email: Switzerland. 1 This article is protected by copyright. All rights reserved. This article isprotected rights by All copyright. reserved. 10.1111/plb.12850 doi: leaddifferences to version between this cite Please of andthe article Record. Version this as been and typesetting, through which may proofreading thecopyediting, process, pagination This article undergone has for buthas and accepted been not review publication full peer Mario Coiro flowers: anexample from the epidermis Nymphaeaceae the floral of andmicromorphology pollinator historical the selectionsignal on Disentangling of &Coiro BaroneLumaga theFloral Nymphaeaceae in phylogeny epidermis and Z-X : Ren Editor typeArticle : Research Paper MR. MARIO COIRO (Orcid : ID 0000-0002-0113-0320)

Accepted– – Article DepartmentBiology, of Botanico,II", diNapoli "Federico Orto degliI- Studi Università Botany, Departmentand Evolutionary of Systematic ZollikerstrasseZurich,University of 1 , MariaBaroneLumaga Rosaria 2

species,ascould homolog beinterpreted which strategiespollination led among tomacromorphological have similarities these flowers of the presents scented flowers pollinated night-blooming, by beetles. Suchsimilar scarab This article is protected by copyright. All rights reserved. This article isprotected rights by All copyright. reserved. quite varied.genus The speciesangiosperms. Among the ofand family, strategies structures floral this pollination are - The family NymphaeaceaeANA-grade oftheamong most diversity the includes Abstract epidermis, with their in present - different showthatthenight-blooming Our flowers specializations results consensusapproachesreconstructionancestral network and state phylogenies. onfixed cruziana of used the stamen epidermis TEM toobserve in papillate-conical ultrastructure of Moreover,we inpollinator attraction.potential homologous ofthe nature structures involved contrasting biology characters pollination toidentify the themain organs floral and of the Nymphaeaceaespecies - for employed We of seven the epidermis with floral of SEM phylogeneticabout these the hypotheses relationships groups. of angiosperms. angiosperms. Acceptedthe Nymphaeales views inthe offloral thatdiverges themostsimplistic evolution from they inthesophistication anatomical a flowers of indicate degree in basisofcantharophilous Nymphaeaceae, the promiseofa and Moreover, anatomy floral phylogenetic marker. show as an independent of- evolution scarab to These in point pollination andparallel results due selection. to pollinator convergence reflect relationshipsThe tendto phylogenetic more epidermal characters than identifyNymphaeales.the presence cells the conical-papillate inthe order first for time of Article . We thentested. We thephylogenetic ofthese distribution traits using or ecological both Victoria cruziana Victoria , as well assubgenera presenting the most elaborate presentingmost the floral anatomy. also We ie s orconvergences different baseds on Lotos Lotos and Hydrocallis

Nymphaea Victoria , micromorphologyin have petal in correlatewith shifts been 2012), and shownto pollinator shifts scalestaxonomical etwell(Costa al.2017)as as among closely (Ojeda related species et al. large modeat2009).associated withpollination Such have traits showntobebeen pollinators hasbeenshown inthe first interaction side abaxial importance cells oftheconical-papillate inthe onthe petals with of important roleintheinteraction between pollinators and For flowers. example, the al. 2011).haveto shown Indeed, an hasand petal been function epidermis themorphology of attractions themicromorphology is floral ofthe (Whitney organs, theperianth et particularly 2004). One importantpollinator morphologywhich offloral aspect is associated with vectors being traditionallyassociated withconvergent et “syndromes” (Fenster floral al. morphology strategies, pollination with theevolution islinked of differentwith pollinations ofthe angiosperms. Flowers stunningare feature evolution most anddiverse offloral the The epidermis This article is protected by copyright. All rights reserved. This article isprotected rights by All copyright. reserved. inflorescence oflarge incase and/or pseudanthia can also traps) with affect the interaction Acceptedbeenof other the focused the micromorphology onthepetals, (or parts flower the of studies haveproduction Even of ofthese (Marinho scent ifmost et compounds al.2014). & insects 2017; (Moyroud Glover Whitneyal. 2011), et involvement inthe as as by well their interaction by meanseffects, ofphysical withlight, grip temperature i.e.interaction or of ofthemicromorphological petals attractionlinked withpollinator have traits or been Articleconical-papillate hydropotes,Cuticle, flower cells, morphology, Key words:

an entire biogeographical entire (Ojeda region et andother al.2016). This mixta mutants of Antirrhinum majus Nymphaeaceae, L. (Whitney et al. secretory This article is protected by copyright. All rights reserved. This article isprotected rights by All copyright. reserved. five in is divided genera:the early-diverging 2008). globe, and diversity present striking ofmorphological levels etThe (Borsch al. family angiosperm family Nymphaeales. around order Members ofthis are the widely distributed ofthemost diversityThe (waterlilies) includes family early Nymphaeaceae ofthe divergent staminodes have investigated notbeen sofar. 2009). However, such interaction and themicromorphological basisof inthe stamens involved inthe attraction and 2010; (Endress theinteractionThien withpollinators et al. eudicots monocots,the are inner ormost also tepals as and staminodes thestamens aswell (Endress 2011), differentiation where the organ between lessclear than is series inthe differentIn so- the (Bröderbauer et pollinators al.2013). Nymphaea Wall. subdivided intoaborealcladesubdivided (subgenus the palaeotropics (subsgenus onedistributed in night-bloomingsubgeneraare with and flowers vicariantly species include which previously includes thespecies in the segregated genus subgenus(subgenus pantropicalTheincludewith one flowers, other species two subgenera with day-blooming by (Fig tips ofthe and carpel extensions it’s 1) by staminodesclosed (paracarpels). inner subgenera of The ecology pollination of theNymphaeaceae interesting Two presents patterns. vicariant Bernhardt 2000), that scarab beetles use as brooding sites. InBernhardt beetles 2000), as thatscarab brooding sites. use AcceptedKnudsen 2003). tobelargegroups inthese flowers tend The ( blossoms Chamber and are of the tribe Cyclocephalini pollinated by (Coleoptera, beetles (Ervik Scarabeidae) (Maia ofscent 2014)bloomingproduce et compounds which flowers amount a al. significant Article , the closelyannuals related L. . Nymphaea This last genus includes most of the speciesNymphaeaceae, of i the This last genusincludes ofthe and most it Brachyceras (subg. Lotos Hydrocallis ) andrestricted to theother) (subgenusAustralia Victoria ) and theneotropics another (subgenus in Nymphaea Lindleyand and Nuphar Nuphar Lotos ) andclades. tropical four Two of these Sibth. &Sibth. Sm ) andgenus the Euryale called“early diverging angiosperms” Victoria, Ondinea Salisb. ., the small genus Victoria , the chamber isformed ) and the largeand genus .

Hydrocallis present night- Anecphya sensu Barclaya

). In s & , This article is protected by copyright. All rights reserved. This article isprotected rights by All copyright. reserved. subgenera subg. chloroplast markers retrievedclade a including ethypothesis 2008). al. 2007, Borsch et (Borsch al. was lacking fast-evolving Analysis of Nuphar On the other subgenera hand, theother of Knudsen 2003). (Ervik blooming would implyhomologya ofthe potential syndromes pollination in supported themonophyly ofthe tropical of subgenera monophylygenus ofthe analysis molecular of sampling data. taxon When became enoughextensive toallowthe emergedimproved by group onthe whichphylogeny have views ofthis thenew from the understanding of strategieshas pollination ofthe been evolution inthe Nymphaeaceae cuppreventsOuret (Thien escaping al.2009). stigmatic from theliquid-filled insects of characters from leaf architecture seem to present (Taylor strongcharactersfrom a architecture signal phylogenetic leaf to seem haveelevated of homoplasy, retrieved levels Indeed,especially characters. infloral if The previous analyses investigatingof morphological theevolution characters inthefamily 2017). polyphyletic support a2011), monophylyNymphaea of et (Borsch al. a full chloroplast sequences support between cladeand the such 2014),number manyfloral floral and oforgans, (i.e. size characters colour) floral are strongly

Accepted ArticleNymphaea, Hydrocallis & are mostly pollinated by diurnal pollinators. Inare mostly subgenera pollinated by diurnal pollinators. Knudsen an 2003).If extremely densely analysis ofthe sampled trnT-trnF spacer did Nymphaea Hydrocallis Nymphaea, the stamens curve on top of the stigmatic cup, and cup, curve asa act the stamens slippery that ontopofthe surface stigmatic and , and ain theseconservatism scenario lineages, and of niche pollination Lotos Lotos and with Nymphaea Lotos, Lotos, Victoria (Lohnewhile different et 2007), al. and gene taxon sampling Victoria thechamber ismostlyformed stamens by erect to be tested, it becamesuch for clear that support to betested, it and related tothe tropical(Grünstäudl etal. Nymphaeas Euryale Nymphaea Nymphaea Euryale clade (Borsch et al. 2008). Such scenarioset 2008). clade (Borsch al. as well as members of thegenusas well as members Nymphaea plus Victoria , and the sister relationship, and the Brachyceras Brachyceras Victoria as sister to and thenight- and Nymphaea (E Anecphya rvik & rvik a & Gee

ecologies. two competing Using these we(Fig. data, test will hypotheses 1F): sample ofmembersfamilyfrom different ofthe clades pollination and withcontrasting Ouraims study family. towards point pollinator attractionpollination astrategies could parallel of inthe evolution the other hand, a different substantially organsbasis for the micromorphologial in involved On ofhomology strengthen strategies these pollination thehypothesis species. between the of This article is protected by copyright. All rights reserved. This article isprotected rights by All copyright. reserved. Accepted Article structural in similar specializations offer evolutionaryits ofevidence history.anline todisentangle independent The presence of Regardingof ecology thepollination theevolution in the family, morphological data could the family. for andmorphology a selection pollinators degreeevolvability high inthefloral from of of synapomorphiesmain cladesand forrole molecular suggests a the supported strong by data, molecular 2008). et signal al. (Borsch therecognitiongenuineThis hinders of mode,linked withpollination generate and whichstrongly a withthe signal conflicts 2) 1)

hypothesis,expect the large, we flowers of scented anatomicalUndersimilar inspecies this with similar specialization pollinators. epidermisThe by determined is structure the strategy, floral pollination of with phylogenetic signal. that theanatomical present should floral characters astrong ofthe epidermis very anatomicalcharacters similar tothelarge,flowers of cantarophilous hypothesis we expect that the small, cleistogamoushypothesis the small, flowersexpect we that of anatomical this speciesUnder strategies.pollination traits inclose with contrasting epidermisThe phylogenetically is structure the floral conserved, of withsimilar to characterize theepidermis characterize floral ofthe organs ina Nymphaeaceae the in Victoria and thenight blooming Victoria and thenight-blooming Nymphaea Nymphaea Euryale Euryale should present should could Victoria , and This article is protected by copyright. All rights reserved. This article isprotected rights by All copyright. reserved. Nymphaea Sepals, flowers were petalscollected andstamens of mature from Plant Species and material studied methodsMaterial and NAP Nuphar lutea Nuphar L. (accession number 19965454 0U, IPENL. XX0Z-19965454), 0U, number (accession 19965454 Anthers of Sepals, petals, stamens from collected electron Scanning microscopy (SEM) oftheUniversity(NAP)Zurich (Z) and theHerbarium (Table of S1). Zurich,Gardens Switzerland. of Vouchers atare the Herbarium Neapolitanum deposited IPEN(accession number 20150988, (accession number 20150989 P0G,IPEN(accession number 20150989 samplesofand sepals,werefrom stamens flowers collected petals mature of Further Botaniquefrom Jardin the collection Belgium. deBelgique, National inthe inMeise, N.gigantea paracarpels andcarpel from collected tips CO for 7°C inliquid at least series,graded dehydrated ethanol critical aweek, point-dried ina Acceptedproducing, sameand time, at toxicity used thereagents ofthe Article 2 and coated with approximatelygold. 25nmof -278) and scent production. scent production. potentiallyNymphaeas anatomical with similar topresent correlated specializations, ) (XX-0- , Nymphaea gigantea Nymphaea Nuphar lutea Nuphar (L.) (XX-0- Sm. Victoria cruziana NAP -2730), -2730), (L.) Sm NAP Hook.(subg. Nymphaea caerulea A. D. Orb. (XX-0- , and respectively petals, stamens,sepals, staminodes, -2731) cultivated-2731) at the ofNaples,Italy. Botanical Garden XX Nymphaea alba Nymphaea -0-STGAL-21500) cultivated Botanical at the XX Victoria cruziana -0-STGAL-2146), and -0-STGAL-2146), Anecphya NAP Th Savigny(subg. , e fixation with ethanol reduces withethanol costs e fixation ) (XX-0- N. caeruleaN. -2732), stamens were collected from were fixed in ethanol were at 50% fixed 4- ex Euryale ferox BR cellent samples for SEM Nymphaea alba Nymphaea , and stamens of -19662012) were-19662012) obtained Victoria cruziana Brachyceras Salisb. Salisb. Nymphaealotus ) (XX-0- ) L. (subgen.

Dissected anthers of electronTransmission microscopy (TEM) V.cruzianaN.gigantea, scanningaccelerating microscopeelectron at 25kV an voltage of ( Specimens wereapproximately examined using 200 a 4nmofplatinum. FEI-Quanta This article is protected by copyright. All rights reserved. This article isprotected rights by All copyright. reserved. carpelstaminodes, stamens, and from collected tips observation. and stamens Sepals, collectedfrom petals buffer in1% post-fixed (w/v) at at4°C, pH7.2overnight OsO %, dehydrated in a graded ethanol series, critical point-dried in liquid CO inliquid %,graded dehydratedpoint-dried ethanol ina series, critical from Borsch et al. (2008),Taylor et al. (2015) and Coiro Coiro and (2015) al. et (2008),Taylor al. et Borsch from taken characters with characters our of integration through matrices of series a then generated We 1). (Appendix produced we data epidermal the from characters three coded We dissection and Signal congruence morphological Italy.FedericoII,Naples, Microscopy ofthe of Centre the Services Metrological University ofNaples of Advanced at anaccelerating of80 voltage kVLaboratory Measures and inElectron Confocal at of the in lead citrateanda examined using electron FEI/ 2088transmission EM microscope Philips 200 meshuncoated uranyl forgrids, 8min copper 12minin stained post-stained for acetate, cut Supernova at70nmusing Sections ultramicrotome. a were collected Reichert-Jung on dehydratedan series. ethanol through embedded wereThey then inSpurr’s resin andsections and the support for ecological groupings (i.e. groupings Acceptedecological for support the and decrease, should datasets combined the in signal tree-like of amount ecology,the pollination reflects flower the of anatomy the if that WeTable S2. in predict explained are combinations Article V. cruziana ) or with a Zeiss) or witha Supra 50VP at anaccelerating 15kV. voltage of were (v/v) glutaraldehyde in3% phosphate in0.1M fixed Victoria Euryale ferox Nympheaea lotus plus the night-blooming Nymphaeas) Nymphaeas) night-blooming the plus & 4 overnight at 4°C, and4°C, overnight at Barone Lumaga (2013); the the (2013); Lumaga Barone were placed in ethanol 50 wereplaced N.caerulea, N.alba, N.alba, N.caerulea, and sepals,and petals, 2 and coatedwith burnin, we generatedburnin, we 50% consensus and both trees conducted 1’000’00 for ratedistributed variation. Two runs(including chains werechains) offour cold three 2012). Matrices analyzed were using acrossgamma- rates and bothequal characters etal. (Ronquist usingThe were MrBayes v3.2.6 Inference conducted Bayesiananalysis (BI) 2006) showingall splits with >0.10posterior probability. (Huson 4 v SplitsTree using generated were replicates bootstrap the of networks This article is protected by copyright. All rights reserved. This article isprotected rights by All copyright. reserved. approachtrees well as as(Denk (Coiroet asplit-network al. 2017) approach Parsimony Maximum bootstrapInferences using Bayesian replicates and posterior Nymphaeales conflictingand thepresence signals, of we employedconsensus-network a To testthe congruence ourcharacters morphological inthe with of other characters would indicate a congruence between anatomical traits and historical signal. analyses molecular by supported clades for support stronger a and tree-likeness in increase prediction. first this of control” “positive a as 2015) al. et (Taylor homoplasy of degree high a present which data, pollen the included We increase. should uncertainties. et the al.2002) matrices onversions in of all ambiguous with characters transformed with >0.10posterior probability.SplitsTree usedtocalculate Delta scoresv 4was (Holland Accepted“as the was using performed analysis Bootstrap swapping. branch Tree-Bisection-Reconnection and taxa new of 10 beta 4.0 addition random PAUP* using replicates, Article 100 for performed using was search Heuristic 2003). conducted (Swofford were analyses (MP) Parsimony Maximum The 0 generations,0 every sampling 1000. 25% as thefirst discarding After - is” option for addition of the taxa and 1000 replicates. 1000 and taxa the of addition for option is” a consensus network showing all splits showingconsensus all splits network & Grimm 2009). 2009). Grimm On the other hand, an an hand, other the On Consensus Consensus & Bryant This article is protected by copyright. All rights reserved. This article isprotected rights by All copyright. reserved. Euryale etZini al.2008; tocode et al.2017) epidermalgeneric characters for at the level We as used well our ascompiled fromobservations data theliterature (Endress 2008; Warner Ancestral character state reconstruction Parsimony criterion using Mesquite(Maddison thesoftware Nymphaea. (Borsch topology eta 2008); al. tree represents the third inwhich a tree withamonophyletic the conflictingfirst themolecular in topologyNymphaeales: of the represents signal data the of ourcharactersthe homoplasy onthree and different distribution representing topologies sister tothe night-blooming Hydrocallis surfacesthese(Table organs was for recorded 1 as fromof ofthe well an connective asinformation connectivetip apical extension (tip), givenof thestamens Nymphaeaceae nature in the laminar species insome and thepresence stomata wererecorded ofabaxial and surfaces adaxial both sepal, and staminodes; of petals For the studiedepidermis, charactersspecies of absence hydropotes and presence or of Terminology represents atree inwhich Carpenter Bhushan(2005)Koch, and Accepted to Carpenter (2006). Article and These dataThese an a were reconstruction with ancestral analysis used state in and Nuphar, Brachyceras Brachyceras at thefor familial level level the subgeneric for Cabombaceae, and Victoria Nymphaea, Nymphaeas Nymphaeas plus Anecphya plus & with subgenus with subgenus Euryale (Lohne et al. 2007). 2007). al. et (Lohne Barthlott (2008); hydropotes terminology accordingBarthlott (2008); hydropotes terminology (Borsch et 2011)(Borsch al. ). are sister tothe tropical Epidermis terminology to according Nymphaea & Maddison 2017). Maddison 2017). We tested ; th sister Victoria e second topologye second to Nymphaea Nymphaea Lotos Lotos plus plus Euryale plus Victoria, clade are cells with lightly wavy anticlinal walls. Stomata and hydropotes are absent (Fig. and Stomata cells lightly walls. hydropotes2A with absent anticlinal are wavy The feroxEuryale the distal margin, bud. the areascovered in in thatwere patches. white between green patches and along sepaloid petaloid latterThe are distributed This article is protected by copyright. All rights reserved. This article isprotected rights by All copyright. reserved. The stamensurface in lutea Nuphar Results https://figshare.com/s/a74f2a7c92fd366805e7. All matrices availableFigShare and trees are on at Data availability In Victoria cruziana (Figs. 2E,F). stamen surface mostly withstomata base shows conical-papillate atconcentrated cells, the The 2D).The sparse petal isodiametrical presents surface domed and hydropotes(Fig. cells 2C). absence stomata(Fig. of andfrom theapparent for the for abaxial the aresparse, density ofhydropotes, which quite surface differstomentose, and adaxial fully trichomes.The covered multicellular inuniseriate areasappear stomata(Fig.densely tobe bears numerous 2B).Some andcovered inhydropotes

AcceptedVictoria cruziana Article ab axial surface of the sepals surface axial the in of

(L.) Sm. (L.) Sm. Salisb. A.D.Orb. A.D.Orb. the abaxial sepal surface theabaxial presents amacromorphological differentiation Nuphar lutea Nuphar shows tetragonal-elongated, slightly convex pavement showstetragonal-elongated, convex slightly Euryale presentstetragonal tabular pavement cells. It cells. tabular presentstetragonal is pavement In the sepaloid theepidermis areas the ).

adaxial petaladaxial surfaces (Figs. 3C,D pavementpetals cells, shareconvex sparse stomata tetragonal, are hydropotes, on absent cell wall (about 5μm) Under cells presents thick with theconnectivethe stamens epidermal TEM observations of stomata. anticlinalepidermis devoid polygonal ofbothhydropotes walls, cells, with exposed and of(Fig.stamens are hydropotes Carpel arealso3I). devoid show although stomata tip present polygonal,The of andand epidermis, hydropotes tips ridged papillate stomata are absent. Paracarpels papillate, 3G,H). show and ridgedare stomata present cells; hydropotes (Figs. stomata arefertile share sterile and present. External polygonal, stamens with epidermis rugulatecells (Figs.3 andabaxial surfacesThe adaxial tetragonal, convex, with staminodes showon epidermis osmiophilic material3L osmiophilic (Fig. dropletsmarked and nm)bounded athickcuticle osmiophilic presents bysmall by (250-600 This article is protected by copyright. All rights reserved. This article isprotected rights by All copyright. reserved. stomata and hydropotes (Fig. micromorphology:similar they showtetragonal pavement cells,convex elongated, sparse present. sepal sepal petaloid areasabaxial The and surface theadaxial appear tohavea In areas,proximal stomata (Fig.3A). areof hydropotes trichomes the uniseriate andsparse ofappears tetragonal composed pavement cells, an tabular tobe with extremely cover dense Nymphaea alba Nymphaea alba Subepidermalsurroundedamyloplasts nuclei cells (Fig. by show Accepted showsanomocyticit stomata and anticlinalpossessessparse hydropotes walls well evident; pavement cells. tabular sepalAbaxial cells, the surfaces with polygonal tetragonal present to Article

shows

E, . total thickness The F), occasionally F), and are sparse cells papillate hydropotes present; little differen ). 3B

). ). and surfaces adaxial and interior Abaxial inexterior both ).

tia tion between organstion ofdifferent regards with series to at the papilla thepapilla is

about 12μm (Fig.about 12μm 3K ). thick cellThe wall is 3J). 3J). N.gigantea, N.gigantea, shapein connective ofthe walls more anticlinal cells, pavement irregular and withcurved and connective cell and thesame type This article is protected by copyright. All rights reserved. This article isprotected rights by All copyright. reserved. In Nymphaea gigantea 4E). topetalquite similar epidermis, (Figs. and cells more inthe former polygonal epidermis inthelatter; respect cellular with to withmore shape, cells elongated tetragonal (Figs. D). 4C, present stamens differentiation alight The between connective and thecae appear Stomata lightlyare absent,anticlinal and tobe quite wavy walls. sparse hydropotes differentiationtetragonal-elongated with between cells present twosurfaces: the they both cells; and4B are stomata hydropotes quite sparse (Fig. (Fig.4A). pavement sepalconvex slightlyThe surface adaxial showstetragonal-elongated, In Nymphaea caerulea (Fig. 5E). connectiveabsent, andareabaxial present hydropotes and theapical onthe extension only latter; the cellularthe same theadaxial sepal are of Stomata and surface. type thepetals is connective, apical thecae, the including protrusion,and the withmore cells elongated inthe surfaceshydropotes D) 5C, Stamen are differentiated are always present(Figs. between the petal andadaxial surfaces, areStomata abaxial petal while surfaces 5B). on absent (Fig. tetragonal-elongated, slightly multipapillate convex boththe in cells thatare also present cells. and are Stomata also hydropotes present sepaladaxial The (Fig.5A). surfacepresents Nymphaea giganteaNymphaea Accepted caerulea Nymphaea Article where of thecellsmore theoutline marked. appears Savigny Savigny Hook. Hook. , the abaxial sepal abaxial presents tetragonal polygonal surface, the to tabular (Fig. 4F ) the stamen presents the same stamen differentiation presents ) the between thecae as

N.caerulea . There isalight differentiation inthe ). petals don’tThe great a show

The sepal abaxial in surface hydropotes,cells(Fig. polygonal and they5 present multipapillate particularlyon theconnective concentrated tip. carpellary ofstomataThe or are devoid tip connective K), and where onallthethe abaxial are side(Figs. they are epidermis, 5J, present Hydropotesbase ofthe structure, canmultipapillate. andfound cellspavement apex on be are polygonal are concentrated and side5I). abaxial ofthe (Fig. onthe onthe Stomata stamen, very differentiation little abaxial. from the The cells pavement are tetragonal-elongate to Hy tetragonal-elongatedcellssurface multipapillate wavy with anticlinals. ofthe presents petal This article is protected by copyright. All rights reserved. This article isprotected rights by All copyright. reserved. the Taylor MP themost in et result al.(2015) trees. These differ trees between inthe relationships from Borschtrimmed matrix ofBorsch (2008)and andmatrix (2008) etal. thefused al. et The trees MP from obtained the analysis ofthe matrices are in summarized Table2. The dissection and Signal congruence morphological Nymphaea lotus dense, and faintly stomata and are and hydropotes multipapillate, equal monophyletic ofaddition ourthree inasinglecharacters MP results matrices toboth tree. This tree showsa of our characters results in an increase in signal tree-likeliness in both Matrix 2 and Matrix 4. 2 and Matrix of bothMatrix ourcharacters inanincrease in results insignal tree-likeliness IndexRetention (Table 2). The different scores for Delta the thattheaddiction show matrices generallyshow a by lowerIndex ofhomoplasy level as Consistency shown and thehigher AcceptedN.caerulea Article dropotes are sparse, and stomata rare (Fig. 5H). stomata rare (Fig. and are sparse, 5H). dropotes The surface adaxial presents petals ofthe Nymphaea ). hy Victoria dropotes spa dropotes Nymphaea species, species,

+ Euryale Victoria , with rs e (Fig. 5F). On the adaxial surface thecellse Onthe5F). sepal, (Fig. adaxial are ofthe Nymphaea lotus is sister to issister N.alba N.alba , and Euryale as sister to Nymphaea. , and result in an unresolved consensus. inanunresolvedresult consensus. , and The presents are polygonal Stomata cells. tabular N.lotus N.lotus The trees from Matrix 2and Matrix 4 The trees from Matrix +( ly N.gigantea N.gigantea sparse (Fig. 5G). (Fig. sparse abaxial The L) .

This article is protected by copyright. All rights reserved. This article isprotected rights by All copyright. reserved. results thecrown increase ina modest inbootstrap supportfor of (2015) situation. doesthis improve not The addition matrices ofourto both threecharacters receives (Fig. support substantial S1 nonode The thatinthe case bootstrap analysis Borsch shows matrix, et ofthe al.(2008) relationship of the sister Nymphaeasandand 52), thefourth),second (53 64for thetropical matrix and rates)forclade and a of probabilitygamma-distributed equal with inthe analysis rates inthe analysis and 0.74 with (2008) matrix presents(2008)marginal matrix a supportfor clade of A However,in the pattern Bayesianresults. similar seen inference is Borsch etthe al. here Nymphaea However,0.5 inthe analysis rates). gamma-distributed with monophyly thesignal for of monophyletic6 (Fig. independently in synapomorphicgenus of the thepavementpresence of ofthe of cells petaloid elongation patches as reconstructed is of our three characters (Figs. 6), as wellof6), as ourthree as differentcharacters (Figs. levels ofhomoplasy(Table The differences tree topologies investigated showsome potential historyreconstructed inthe Ancestral character state reconstruction lotus Euryale Nymphaea lotus Acceptedmonophyly oftropical Article Euryale disappears with the addition of our characters (Tabledisappearsaddition ofour characters with the 3). and is onlyfor marginallyclade a of than stronger a signal (63 and 65) (Fig.S1B and 65) (63 Victoria N.caerulea N.caerulea (T Nymphaea ab . On theother hand, a between. On support for link A) le Nymphaea 3). 3). The addition supportfor ofour characters the the improves ; in thesecond topology, ; elongated evolved state either the has N.caerulea and subg Nymphaea N.gigantea , the monophyly of the genus and the relationship between oftherelationship genus and, themonophyly the ). Nymphaea and

A) , and the addition ofthe charactersfrom addition , and the Tayloret al. inthe topologygenusas reconstructed is the were N.gigantea N.gigantea (53 and 53)as well as for the grouping53)as(53 wellthe and as of for or has been or inthe has lost (0.59 in the analysis with equal(0.59 inthe analysis rates with and Euryale and Nymphaea Nymphaea Victoriacruziana Victoria Victoria Victoria ( - and Euryale (73 for the 0.67 posterior S3 Nymphaea ). The Victoria and clade ambiguous (Fig. 6D); in the third topology,ambiguous third 6D); inthe (Fig. papillate simple state of the derivesa state multipapillate from (Fig. and it’s the in lost the papillate after the ofsubg states originate split inthepapillae second6B); being topology, for synapomorphic Nymphaeas thetropical (Fig. This article is protected by copyright. All rights reserved. This article isprotected rights by All copyright. reserved. (Fig.6C Victoria papillaethe synapomorphicsimple being for the Th organ same pattern followsthe series exact petal as cell the elongation. (2008, sepaloid 2009). surface However, this insome differs ways between the outer surface sepaloid asWarneralready surfaces, andnoted by et sepal theother al. ofspecies.between investigated Allspecies a groups differentiation different present between organ studied adegree epidermis seriesbetweenpattern clear of show differentiation the of Theinfluence morphology the evolution. epidermis floral factors onits and ofhistorical of and thefunctionalOur the waterlilies data epidermisfloral the offer in role into of insights Discussion Accepted alsoleaf These2006). share (Carpenter surfaces evident twospecies differentiation an and givenparallelism specialized of these that cells the distribution with inthe leaves, groupsinteresting ofspeciessepaloidthese patches tepals two presents outer in ofthe Nymphaea, Article e and papillae simple multiple are as reconstructed independent topology, thefirst in with Euryale , where ); in the third topology, inthe third evolves beforeelongated ofsubg thesplit state the where it is not. This difference in the distribution of the hydropotes between of Thisdifference thehydropoteswhere isnot. the it inthe distribution represent the only taxa in the Nymphaeales to have adaxial hydropotes onthe adaxial have the onlyrepresent inthe Nymphaeales taxa to it is densely covered in trichomes and hydropotes, and ofandcoveredspecies the hydropotes, is densely intrichomes Victoria - Euryale clade (Fig. 6E). 6E). clade (Fig. The epidermal differentiation between 6F ). ). Victoria-Euryale Nymphaea , but the ancestral stateis , butthe ancestral clade and the multiple multiple the clade and Victoria Euryale and Victoria Nymphaea and Euryale

This article is protected by copyright. All rights reserved. This article isprotected rights by All copyright. reserved. reported from has onlypapillate quite rare and cell cells. amongbeen angiosperms, latter type basal This is cellspresenting andpetals conical- stamens presentingcells, presenting tabular domed withsepalsbetween cell theepidermis typesthe different the in present of organ series, the petalIn epidermis. three tropical the epidermal in cells typesalso tetragonal-elongated share inthe different They all series. organ Nymphaea gardneriana This epidermalalso in been type has showntobe present of the sepaloid surface adaxial the outerpresent tepals. theexclusion all organs, in of with The flowers of the species of thegenusThe the flowersspecies of beingwith radially arrangedparacarpels. ofstamens present onthe epidermis and folds extremelythe differentiation with cells maximum, reaches its conical-papillate specialized Accepted in that wouldalso take of consideration floral into distribution regulator expression thespatial (2008;2009) underline observed, and the necessity studygeneexpression an approachof to & 2010; confirmal. Soltis, 2010). YooetSoltis Our et what al. Warner observations Theissen 2007),& of waterlilies gene as Melzer, reported by inthe studies expression (Yoo, ofSaedler, of& expression thefloral regulators 2001; of system the(A)BCDE (Theissen the socalledexpand “fading borders” model(Buz controlled byan interaction of internal factors.environmental went onto and Thismodel the evolution, toparticular ofsepalness the features were notfixed and organs, petalness but inanearly groupal. of Modelfor 2008,2009).The that, stage proposedpredicts this Mosaic group,peculiarities like thepresence of this ofdifferent et organs (Warner insingle patches The organ study inthe waterlilies determination of identity have focused on the mostly Differentiation hierarchies the in waterlily flower Article Austrobaileya from subgenus from among ANA-grade taxa (EndressIn 1983). among taxa ANA-grade Nymphaea Nymphaea Hydrocallis Hydrocallis Nymphaea Nymphaea goal., 2004),thatarea postulateda et wide lack between clear differentiation the species, a cell multipapillate type is (Zini et al 2017). (Zini et al2017). Nymphaea amazonum Nymphaea amazonum Victoria cruziana Victoria and

and thestamens in water asreflected i.e.inthemacromorphological lilies, outer “gap” between staminodes the moreorgans is closely ofthe withrespect theother series single to species ofthe determined speciesleast the regardsepidermal identity with It this ofthe to morphology. that in seems Euryale ferox This article is protected by copyright. All rights reserved. This article isprotected rights by All copyright. reserved. paracarpels of the stamensThe and epidermis particularly presence the papillate-conical inthe in of Functional the aspect of epidermis in Nymphaeaceae at could of the base cell be differentiation types. ofthe differential flowers ofexpression these of oftheepidermis genes more the complex inthe genesof MYB theR2R3 inthe genome of epidermis ofapetalous flowers et copies (Diof al.2009). multiple Given Stilio thepresence the development (Noda cells indifferent et ofconical models al.1994), staminal including MYB proteins thedifferentiation These in of epidermis. proteins floral been have involved in stimulating Apotentially question. avenue promising for role of research involves R2R3 the & 2010; Soltis, Yooet etand2006; Kim al.2010) al.,2004;Yoo,Soltis isaninteresting genespoorly (Geuten inthe waterlilies expression ofal. differentiated theMADS-box et the single organ organs. The series strong differentiation of in the similar inshape,similar are differentas quite to the compared conical- their structure regard to with petal identity (Irish 2009). petal identity 2009). (Irish The of epidermal cells et al.1981; Whitney 2011), et al., authors usedeven as by various amarker for been and has 1975). Acceptedthe sophisticated biology pollination genus the(Valla of Article A papillate-conical epidermis a is commonpapillate-conical feature (Kay flowers inmany angiosperm , on the other hand,of regulation, a, ontheother sophisticated demonstrate system more at Victoria cruziana Victoria . How this differentiation is possible, given. Howdifferentiationpossible, is this wide and the relatively and their peculiarultrastructure suggest to seems with alink Victoria cruziana Nuphar advenaNuphar Victoria & , however,if extremely even (Brockington et al.(Brockington 2013),the et Cirino 1972 Cirino Victoria cruziana Victoria : Prance & Arias and This article is protected by copyright. All rights reserved. This article isprotected rights by All copyright. reserved. type.with neutral if stainingandEven red olfactory analyses (Valla large nuclei. This organizationancould epidermal indicate a secretory such role potential for and droplets plastoglobuliunderlying with lipid plastids andstarchgranules, presents tissue cuticle surface. deposits, andthe with osmiophilic The apresent vacuole epidermal cells substancesto bechannelled, inthechannels arevisible and deposited onthe osmiophilic and could Moreover, represent terpenoids lipids, orothercompounds. volatile cuticle appear the cell that wall isabnormally appears thick, it compounds, toosmiophilic tobe permeable from TEM images. ofthethat, even thickening ifthe ultrastructural shows The observation as evident itself) cell centralwall part ofthe wall (i.e. ofthe thickening a dome-shaped solid by2006), thickening umbo-like an appears the unusual tobe external formed of periclinal represents ofthe body cellular extension atrichome-like (Kayet al.1981;Effmert 2005, et al. papillate cellsangiosperms. ofother cells normal in conical-papillate papilla, that The Accepted investigation. specific troughis beyondemission the scope analysis current time, of the of scent & 1986). Whitten Awhich detailed would involve emission, an more analysis organ- ofscent Kolosovaosmophoric (BaudinoStern, et Curry tissue et al.,2001; al.2007;Bell2009; et al. our data are sufficientidentify theconnective stamens of to of the ultrastructural thatwith do notthink among we orhistological characters angiosperms, basal inner et al.2017).scarcity tepal (Zini Given epidermis the correlating ofdata emission scent a staining similar role ofthe tothe behaviour inner speculated tepals,functional and about the least during investigations first offlowering, have night stamenspresentlater the shownthat suggest Article ed that stamens and paracarpelsat stamens and emission, involved inscent are that notparticularly Victoria cruziana & Cirino 1972) originally 1972) Cirino as an s presencea epidermis ofmultipapillate for could clade including besynapomorphic all represent for genuine The synapomorphies clades some ofthe Nymphaeaceae. ofthe Our of ourcharacters character analyses indeed reconstruction some could indicate that anatomy the organs floral of andthe organ showthat Moreover,the differences and number. 4 between 2,3, matrices typepresentBorschetcharacters such some inthe of the al.(2008) as in pollination matrix, overcome Such signalecologicalpresent signal the to manages larger matrices. in probably presents complements thesignal which and improves arather strong phylogenetic signal, 2002). These allshowthattheanatomythe floral results of Nymphaeaceae organs the in including andclade BIboth MP 5). (Figure Our reduce a characters thesupport night-blooming also for present indicatingDelta lower scores, “tree higher groupingNymphaeas tropical ofthe (subgenera support for the groupingsupport forof the This article is protected by copyright. All rights reserved. This article isprotected rights by All copyright. reserved. Accepted Articleby Coiro et and Taylor al.(2015) identified morphologicalwith the extensive et ofBorsch al.(2008), characters and matrix the conclusions, phylogenetic withourcharacters combined When signal. presenting tree-like these Ourwellcharacter supports analyses signal as dissection as reconstruction our Nymphaea caerulea contrasting ecologies, between pollination i.e. characters.hand, other similar anatomicalcharacters are Onthe species shared with by waterlilies.with shared ecologies anatomicalpollination The donotshare particular species inthe anatomicalour two hypotheses Our specialization about results allowustotest homoplasiesTesting morphology in floral Victoria and . . Nymphaea lotus. Nymphaea Victoria is less labile compared to pollen characters (Taylor et al. 2015). lesslabilecompared characters 2015). topollen al. (Taylor et & Barone Lumaga (2013), our characters increaseBaroneLumaga(2013),characters the our and Moreover, the matrices Moreover, ourcharacters thatinclude Euryale, Victoria Victoria Lotos, Anecphya Lotos, the monophyly of -l ikeness” of et thesignal al. (Holland and Euryale, and Nymphaea Nymphaea or Brachyceras Nymphaea lotus Nymphaea and the ) using using and well as insubgenus This article is protected by copyright. All rights reserved. This article isprotected rights by All copyright. reserved. tropical subgenera of Zurich. Zurich. equipmentImage maintained by theCenterfor Microscopy and University Analysis, of help thecollection with of specimens study. usedinthis Imaging was with performed staffZurich)Manfred and Garden(Botanical Knabe of forBotanicalNaples of Garden the extreme specialization of thefloral in epidermis 2011). Theclassical (Whitney pollination with across link its al. theangiosperms and et representsgiven another thelevel striking synapomorphy, characterthis ofhomoplasy of epidermissuch conical-papillate clade of The topologies. inallthree thank Elke Bellefroid,(Botanic acknowledgesMC Linder for work. supporttothis H.Peter hisfundamental We wouldlike to Acknowledgements family. the in investigation andtraits, genetic intheir evolution these inthe and structural basis of basisrepresent ofpollination strategies apromisingavenue further inNymphaeaceae, and for providediversity key anatomicalNymphaeaceae. insights ofthe the in Nonetheless, they present could still characters ahigher level the ofhomoplasy identified over whole here relatively samplingour taxon Though and isstill that the be should noted it limited, phylogeneticon thegroup 2008). et hypotheses (Borsch al cantarophyly 2003) independently (Krell et each al., from byas other, suggested some ofthe Accepted Articlethe beetle-pollinated Hydrocallis Nymphaea lotus, lotus, Nymphaea Nymphaea, al (Ziniand isreconstructed it etbe al.2017) ancestral to to since it Garden of Belgium, Meise), PeterBelgium, Rene Gardenand Enz, of Gerber strengthens thehypothesisstrengthens species that adapted to such is present in all thesubgenera present in as investigatedhere Victoria cruziana , and thedifferences with Euryale Euryale and Victoria Victoria Signaling & Signaling Behavior, Hugueney P.volatilecompounds of by emission and Cells. (2007) petal Production BergougnouxBaudino Caissard J.C., S., V., F., Jullien Cock J.M., G., J.-L., Magnard Scalliet basalgenomics comparative angiosperms for research. P.S., Hong C.W., Ma, dePamphilis Leebens-Mack(2005) FloralgeneJ.H. fromresources Treves Zhang Bliss B.J., X., SchlarbaumTanksley R., S.E., Oppenheimer S.D., D.G., Soltis L.A.,MuellerLandherr L.L., Yi Kim S., Buzgo Hu, M., Yoo Frohlich M.W., M.-J., Perl- Albert V.A., Farmerie D.E., Carlson J.E., Soltis W.G., Wall P.K.,Ilut Solow D.C., T.M., Literaturecited manuscript. thank the editoranonymous and two helping for reviewers improve todramatically the Guy for stylistic Atchison thoroughand isthanked commentsWe linguistic on issues. also This article is protected by copyright. All rights reserved. This article isprotected rights by All copyright. reserved. factors. K., Martin Baumann Bradley M., K., Perez-Rodriguez D.,Venail Bailey J., P., H., Koes Jin R.,Roberts manuscript and Peter the epidermal for Endress about discussions morphology of We wouldalso toacknowledge like versionfor of this comments Paula a Rudall on previous AcceptedBorsch T.,Löhne Mbaye C., M.S., Wiersema (2011) J.H. Towards speciescomplete a tree of 369-387. cladesOrchidinae (Orchidaceae). ofsubtribe Comparative micromorphology nectarless ofnectariferous and inselected spurs labellar Bell P.J., Rudall Hawkins J.A., Roberts D.L., A.K., Bateman M.S., Box (2009) R.M. Article Development , Development C. (2007) ofcell Control and morphogenesis petal by transcription MYB R2R3 2 134 (6), 525-526. (6), 525-526. , 1691-701. Botanical Journal ofthe Linnean Society,Botanical BMC plant biology,BMC plant 5 , 5. Victoria Plant Plant 160 . , This article is protected by copyright. All rights reserved. This article isprotected rights by All copyright. reserved. Nymphaea water-lilies. trapsgenus ofthe Bröderbauer, D., Weber, A., & Diaz, A. (2013). design oftrapping devices inpollination The evolution, highlights targets potential for thestudy cellular differentiation. of (2012) EvolutionaryL.C.,HilemanB.J. analysis ofthe Glover MIXTAgene family Brockington S.F., Landis R., Alvarez-Fernandez J.B., K., Alcorn Walker R.H., Thomas M.M., Nymphaeales:genomesgenes integratingand morphology. , Borsch T.,Löhne C., Wiersema (2008) Phylogeny in and J.H. evolutionary patterns morphology, and homology. distribution, Carpenter structures(2006) leaf Specialized epidermis ofbasal inthe K.J. angiosperms: Journal ofBotany, Carpenter(2005) basal in architecture Stomatal angiosperms. K.J. evolution and 173. geneticmorphological and studies offloral development. P.S.,Buzgo Soltis D.E., Soltis M., Ma H. (2004) Towardsintegrationcomprehensive of a Society mi (2013) BaroneLumaga M., Coiro M.R. Nymphaeaceae: in evolution Aperture a insights from Microscopy.In Iodide Propidium Staining of MountSamplesLaser-ScanningWhole and Confocal M., Coiro Truernit(2017) E. Xylem Characterization UsingImproved Pseudo-Schiff Coiro M., Chomicki G., G., Chomicki M., Coiro Doyle signalExperimental sensitivity J.A. dissection and method

Acceptedcromorphologicalultrastructural and investigation. Article , 172 30 : shedding further light: shedding further onsubg. (3), 385-397. (3), 385-397. Telopea, (3), 526-540. (3), 526-540. Xylem: Methods andProtocols,Xylem: Methods Arum 92 13 , 1595-615. 1595-615. , (Araceae) isrelated toinsect type. , 193-217. , American JournalAmerican ofBotany, Brachyceras Springer (New York), 127-132. Grana, and its relationships tothe and relationships its Australian Trends science plant in Botanical Journal of theLinnean of Botanical Journal Taxon, 52 (3), 192-201. (3), 192-201. Molecular biology and biology Molecular 57 , 1052-1081. 93 , 665. , , American 9 (4), 164- (4), composition, emission pattern, emission composition, and of localization floral scent in emission Flowers. Endress P.K. (2001) Flowers Basal inExtant The InferencesAngiosperms and on Ancestral and Development, Endress, P.K. inontogeny of (1990) construction floral Patterns and phylogeny. 103 Endress P.K. (1983) earlyof development floral The Austrobaileya. (Nyctaginaceae). Effmert U.S. Röse Große U., J., ScentBiology ofFloral Emission of ScentEmission Compounds the Flower. within EffmertLocalization B.Synthesis ofBuss(2006) D., and the Piechulla D., U., Rohrbeck controls epidermal shape of cell inflowers Thalictrum. micromorphologyand relationship pollination. its to Costa V.B.S., Chagas R.M.M., Pimentel M.G.S., Alves (2017) G.D., CastroC.C. Petal Endress P.K. (2011) theangiosperms. Evolutionary flowers of diversification in Sciences, Journal ofPlant Endress P.K. (2008). biologygrade Perianth angiosperms. inthe basalextant of Di Stilio Di Stilio V.S., Martin Palaeobotany andPalynology, Denk T., Grimm G.W. (2009) historyThe biogeographic ofbeech trees. This article is protected by copyright. All rights reserved. This article isprotected rights by All copyright. reserved. Accepted Article of angiosperms andGnetales. analyses reaffirmof therelationship offossils andmorphology inthe resolution the potential (4), 481-497. (4), 481-497. International Journal of Plant Sciences,International Journal ofPlant American Journal ofBotany,American 1 ,153-175. ,153-175. C. , 105-124. , Schulfer A.F.,C.F. Connelly (2009) of MIXTA-like2An ortholog 169 BiorXiv, , 844-862. 158 R. , EhrigF., Piechulla B.R., (2005) Kägi Volatile , 83 – 134262. 100 100 92 , 2 , 162 In – N.Dudareva,and E. Pichersky [eds.], Plant Biology,Plant 12. , 1111 New Phytologist, Phytologist, New -1140.

19 Botanische Jahrbücher , 115 Review of of Review 183 Mirabilis jalapa Mirabilis – 122. ,718 International Evolution American – 728.

, This article is protected by copyright. All rights reserved. This article isprotected rights by All copyright. reserved. and genes(2006) Petaloidyand inthe balsaminoid identity genera petal MADS-box GeutenBecker K., P., K.,Kaufmann Caris A., Janssens S., Viaene T., E. G., Smets Theißen Systematics syndromesPollination floraland specialization. Fenster,B., C. Armbruster, W. S., Wilson,P., Dudash, & M.R., D.(2004).Thomson, J. Society, Journal oftheBiological Linnean Ervik F., Knudsen J.T.(2003) Wateryears? faithful 100 million and for lilies partners scarabs: Journal ofBotany, Huson D.H., Bryant phylogenetic distance data. Holland B.R., K.T., Huber Dress A., Moulton V. (2002) structure inpetals. (1981) C.H. Pigment light Stirton distribution, reflectionKayDaoud Q.O.N., H.S., and cell Irish V.F. of petal identity.(2009) Evolution &Molecular Bology Evolution plant surfaces. B.,BhushanKoch Barthlott K., W. (2008) Diversitywettingof structure, morphologyand of 2118. ofaimplications 260-million-year-old duplication. genesangiosperms:diversificationB-function MADS-box evolutionaryfunctional of and in Kim S., Yoo M.J., Albert V.A., P.S., Soltis Farris J.S., D.E. (2004) Phylogeny Soltis and AcceptedN. (2001) KarlsonKolosova Cellular N., and Sherman D., D., Dudareva subcellular Article Marcgravia , 35 Soft Matter,Soft , 375-403. , 375-403. . The Plant The Plant Botanical Journal Society, ofthe Linnean Botanical 98 D. , 370-396. 370-396. , (2006) (2006) networksApplication ofevolutionary phylogenetic studies, in 4 Molecular Bology &Molecular Bology Evolution Journal, , 1943-1963. , 23 (2), 254-267.

47 , 501-18. 80 Journal of experimental of Journal botany, , 539-543. , 539-543. Annual Review Ecology, of Evolution, and American Journal ofBotany, δ Plots: a tool for atool analyzingδ Plots: , 19, 83 2051 , 57-84. , 57-84. – 2059. 2059. 60 , 2517-27. , 2517-27. Impatiens 91 , 2102-

This article is protected by copyright. All rights reserved. This article isprotected rights by All copyright. reserved. flowers. enzyme for ester snapdragon responsible ofthevolatile biosynthesis methylbenzoate in ofS-adenosyl-L-methionine:benzoiclocalization acid carboxyl methyltransferase, the in AfricaMagnoliidae: (Coleoptera: Scarabaeidae: Dynastinae; Nymphaeaceae). Krell F.T., (2003) S. Rhinoceros Porembski beetles R., water pollinate Hirthe G., Seine lilies Sciences,International Journal ofPlant Lower ofEvidence Studies the Comparative Eudicots from andBasal Angiosperms. IrishKramer E.M., V.F. Programs: ofthe Evolution Developmental Petal and (2000) Stamen night-blooming water and putative pollinators. lilies, their with relation flowers. C.Marinho, R., Souza, D.,Barros, C. T. C.,& Teixeira, P. S. Scent (2014). glands inlegume 67-75. (2014) floral of The scent Maia LimaC.T., A.C.D., de NavarroD.M.D.A.F., Chartier Giulietti M., MachadoI.C. A.M., 9 specialized cell-shape controlled by a Myb-related transcriptionspecialized cell-shape by factor. controlled a Myb-related Noda K., Glover 350 Ojeda D.I., Valido FernándezA., de Castro Ortega-Olivencia A.G., J., A., Fuertes-Aguilar SciencesJournal ofPlant revealsa ofpapillose loss evolution conicalcells ofbirdpollination. during the Q. (2012).micromorphology Macaronesian Comparative in (Leguminosae)Lotus ofpetals I.,Ojeda, Santos-Guerra, R.,Marrero, Jaén-Molina, A., Caujapé-Castells, J., &A., CB Cronk, Moyroud E., Glover B.J. (2017)MoyroudB.J. Glover E., attraction. pollinator The physics of Accepted Article, 103 – 354. – 106. Plant physiology,Plant Plant Biology Plant B. , Linstead , , 16 , Nymphaea (1), 215-226. (1), 215-226. 126 173 P. , 956-64. (4), 365-374. (4), 365-374. , Martin C. (1994) Flowercolor(1994) on , Martin intensity C. depends subg. 161 Hydrocallis , S29-S40. , S29-S40. (Nymphaeaceae), New the World New Phytologist, New Nature, Phytochemistry, 369 International Ecotropi ,661

216 –

103 664. ca , , , This article is protected by copyright. All rights reserved. This article isprotected rights by All copyright. reserved. Prance G.T., (1975) Arias J.R. A the study biology floral of of the MacaronesianIslands bird-flowered species. Santos-GuerraCarvalho J.A., evolution on shiftsdrive Pollinator petalA. (2016) epidermal Sowerby (Nymphaeaceae). inference a across choice large and model modelspace. L. Ronquist, F.,Ronquist, Teslenko Bulletin ofthe TorreyBulletin Botanical Club, Stern W.L., Curry K.J., Whitten W.M.(1986). flowers. Stainingglands orchid fragrance in studies. architecture characters: molecular testing and vegetative hypotheses phylogenetic from Taylor D. W., Gee C. T. based lilies Phylogenetic offossil water (2014). onleaf analysis Methods). Version 4. Sinauer Associates, Massachusetts. Sunderland, Swofford(2003) PAUP*.Phylogenetic D.L. UsingAnalysis Other Parsimony(*and Botany (2009) biology Pollination J.H. (ANITA ofbasal angiosperms grade). L.B.,Thien Bernhardt P.,Luo Devall ChenZ.D., M.S., Y.B.,Fan J.H., YuanL.C., Williams Taylor J. Schneider E.L., M.L., Cooper Osborn R.L., lineage. developmentInsights Nymphaeales: character in into angiosperm an evolution in ancient of theangiosperm flower. underlying MolecularTheissen(2007) G., Melzer R. diversification mechanisms origin and Theissen Floral (2001) G., H. Saedler quartets.

Accepted Article M. , Suchard P.Huelsenbeck J. A., (2012) MrBayes efficient 3.2: phylogenetic Bayesian , 96,166 Bulletin ofthe PeabodyBulletin History ofNatural Museum American Journal of Botany, of Journal American – 182. M. , Van Mark Der Annals of Botany, Annals of Acta Amazonica, 102 113 ,1685-1702. ,1685-1702. P. , 288-297. , Ayres D.L.,Darling 100 5 ,109-139. ,109-139. Nature, ,603-619. Biology Letters, Biology M. 409 (2015) structure Pollen and Systematic biology,Systematic , 469 , 55 Victoria amazonica , 89-110. –

A. 12 471. , Höhna , 20160022. , 20160022. American Journal of American 61 S. , Larget, , 539-542. (Poepp.) B. , Liu , Warner K., P.J, Rudall Frohlich M. Nymphaeales. D'Orb.(Nymphaeaceae). This article is protected by copyright. All rights reserved. This article isprotected rights by All copyright. reserved. Valla(1972) D.R. Cirino Biologia J.J., floral delirupé, allow bees to gripallow bees and increase flowers to foraging efficiency. Whitney, L., H.M.,Chittka, Bruce, T. & Glover, J., (2009). B. Conical cells epidermal J. a differentiated perianth. petalness perianth in organs ofwaterlilies:Mosaic the theory evolutionary for anew origin of Warner K., P.J., Rudall Frohlich M.W. (2009). Environmental control ofsepalness and Nuphar advenaNuphar in the floral transcriptome: insights thewater from basalmost oneof angiosperms, the lily Yoo Chanderbali M.-J., A.S., P.S., Soltis Altman N.S., D.E. Soltis (2010)Evolutionary trends 616. (2011) petals have dosomany Why conical epidermal cells? Whitney BennettH.M, K.M.V., L.,B.J. DorlingL., D., Chittka Prince Glover Sandbach M., 953. Divergent WaterLilies: Nymphaealesand Yoo P.S., Soltis M.-J., D.E. Genes Box (2010) Soltis of Floral Expression in MADS Two

Acceptedstudy onepidermal and structural characters (2017) L.M.,Ferrucci organsZini Perianth B.G., M.S. Galati Nymphaeaceae: in comparative 171 Article , 121-146. Taxon, (Nymphaeaceae). 57 , 1096-1109., Darwiniana Journal ofexperimentalbotany, The Plant journal, journal, The Plant W. , 477-500. (2008) Differentiation of perianth organs in Nelumbo . Journal of Plant Research Plant of . Journal . International Journal of Plant Sciences,International Journal ofPlant 64 Victoria cruziana , 687-98. 60 Current Biology , 3559-3574. , 3559-3574. Annals of Botany,Annals of ,

130 , (6),1047 19 108 (11), 948- , 609 – 1060. 1060. – hypothesis 1, closely related genera (Ctips), paracarpels or inner staminodes (Pc), stamens (St), and petals (Pt) are indicated. In F, under hypotheses around the floral anatomy Nymphaeaceae the of In (F). carpels Ethe (Car), carpel tips This article is protected by copyright. All rights reserved. This article isprotected rights by All copyright. reserved. and(C), topologysection 3(D), of about the phylogeny the of Nymphaeaceae plus Cabombaceae (B-D), i.e. topology 1(B), topology 2 Figure1 Figurecaptions pollination ecology. Under hypothesis 2, Figure2 Nymphaeas. convex with cells wavy walls.anticlinal surface surface showing conical-papillate cells; showing tetragonal, convex pavement cells, and hydropotes E, SEM (h); image stamen the of abaxial C,SEM image sepal adaxial of surface showing hydropote D,SEM (h); image epidermis petal of epidermis showing tetragonal, lightly convex pavement cells, sparse stomata (s) and hydropotes (h); Figure3 cells (p), and stoma (s).Scale bars, 50µm (A); 20 µm (B,C); 30 µm (D); 100 µm (E 100 µm (D); 30µm (B,C); 20 µm (A); cells (p), and stoma (s).Scale bars, 50µm showing polygonal, papillate, ridged cells, hydropotes and stomata are absent; I, SEM image fertile of Acceptedhydropotes (h) and stomata (s) are present; H, SEM image fertile of stamen connective surface SEM image external of sterile stamen adaxial surface showing polygonal, papillate ridged cells, staminode adaxial surface showing rugulate surface of andcells papillatestomata cells, ( rugulate, occasionally papillate cells, sparse hydropotes, (s) are stomata present; F,SEM deta sparse hydropotes E, (h); SEM image staminode of abaxial surface showing tetragonal, convex, present; D, SEM image adaxial petal of epidermis showing tetragonal, convex pavement cells and petal abaxial surface showing tetragonal, convex, rugulate cells, sparse hydropotes, stomata (s) ar showing tetragonal elongate, tabular slightlyto convex cells, sparse hydropotes C, SEM (h); image of polygonal tabular cells with hydropotesand stomata (h) SEM B, (s); image adaxial of sepal surf Article . Relationships between the main clades of the angiosperms main (A), molecular hypotheses . . Nuphar lutea Victoria cruziana: (A): A,SEM imagestamen of epidermis showing tetragonal elongated, lightly A,SEM image sepal abaxial of epidermis showing tetragonal to a Euryale flower of F, Euryale ferox Victoria SEM image stamen of epidermisshowing papillate conical and Victoria cruziana Victoria share more similarity with the night-blooming

(B share similar anatomy contrastring but -F ): B, SEM image sepal adaxial of (E), and summary the of main ); 10 µm (F 10 µm s) present;s) G, il ofil ). ace

This article is protected by copyright. All rights reserved. This article isprotected rights by All copyright. reserved. (A-E, G, H secretion Scale(es). 100µm bars, TEM image fertile of stamen wallpapilla cell showing channeled cuticle (cc) and extracell subepidermal cells with plastids with starch granules (sg), droplets lipid (ld) and large nuc wallcell with waved surface a and tangentially sectioned papilla;K, TEM image ferti of image stamen fertile of epidermal cells showing cytoplasm boundedby osmiophilic material (*), thick stamen tipsurface showing polygonal, papillate, ridged cells and two stomataanomocytic (s); J, Figure4 absent. stamen connective surface showing tetragonal to polygonal cells, andhydropote (h), stomata are elongated slightly convex cells and sparse hydropotes (h), stomata areSEM absent; image E, the of hydropotes, stomata are absent; D, SEM image petal surface the adaxial of showing tetragonal- SEM image the petal of abaxial surface showing tetragonal-elongated slightly convex and cells sparse surface showing tetragonal-elongated slightly convex cells, sparse hydropotes (h) and stomata; C, polygonal tabular cells, sparse hydropotes (h) and stomata B,(h); SEM image the of sepal adaxial Figure 5 (F). 10 (E); µm multipapillate cells, stomata (A-D); 50µm are absent.Scale bars, 100 µm lotus tip surface showing multipapillate polygonal cells and hydropote (h), stomata are absent. elongated multipapillated cells, dense hydropotes (h), stomata are absent; E,SEM image stamen ofthe hydropotes(h)and sparse stomata; D, SEM image the of petal adaxial surface showing tetragonal- SEM image the petal of abaxial surface showing tetragonal surface showing tetragonal-elongated multipapillate cells, dense hydropotes (h) and sparse stomata; C polygonal to tabular cells, hydropotes (h) and sparse stomata; B, SEM image the of sepal adaxial showing tetragonal multipapillate cells, sparse hydropotes (h) and stomata; H, SEM image petal the abaxial of surfac (s) and sparse hydropotes G, (h); SEM image the of sepal adaxial surface showing tetragonal- Accepted and rare stomata; I, SEM image the petal of adaxial surface showing tetragonal-elongated Article (F-L Nymphaea gigantea . . ): Nymphaea alba Nymphaea caerulea F, SEM image the of sepal abaxial surface showing polygonal tabular cells, dense stomata – elongated multipapillate cells with wavy walls, anticlinal sparse hydropotes (h)

(A (F -E (A-E ): ): SEM image stamen the of connective surface showing polygonal A, SEM image the of sepal abaxial surface showing tetragonal to ): A, SEM image sepal the of abaxial surface showing tetragonal ); 10 µm (F, 250nm (K); (L 10 µm I, 5µm J); – elongated multipapillate cells, dense ).

le stamenle Nymphaea lei (n); L, lei (n); ular TEM TEM e , tropical This article is protected by copyright. All rights reserved. This article isprotected rights by All copyright. reserved. a topology monophyletic with and the Papillae character (Character 3) ontheusing right Parsimony. is Character reconstruct Figure 6. (L). 10µm (G); (A-D, F, (E); 30 µm 100 (J); µm H,I,K); 50 µm bars, 200 µm and stomata (s); L,SEM image the of carpellary tip showing polygonal multipapillateSca cells. SEMK, image the stamen of adaxial surface showing polygonal multipapillate cells, (h) hydropotes showing polygonal multipapillate cells stomata and and (h), pollen hydropotes grains are visible (p); multipapillated cells, sparse hydropotes (h), rare stomata; SEM J, image abaxial the of stam Accepted signalthe and the support for the monophyly of characters). Only splits present morein than 15% of the replicates are shown. Both the treel Matrixa) 1 (data from et Borsch al. 2008) and b) Matrix 2(data from Borsch et al. 2008plus our S1.Figure Article Nymphaeas (E,F). tropical Nymphaeas (C,D), and a topology with Nymphaea Ancestral reconstruction of Petaloidthe pavement cells character on the(Character left 2) Consensus networks the of bootstrap replicates fromMaximum the Parsimony of analysis clade clearly improve from b. ato Nymphaea

(A ,B), a topology with Nymphaea, Victoria and the Euryale Victoria Victoria as sister theto night-blooming plus and Euryale Euryale clade and the sisteras to the en surface en surface ikeness of ed over le le This article is protected by copyright. All rights reserved. This article isprotected rights by All copyright. reserved. Accepted Article This article is protected by copyright. All rights reserved. This article isprotected rights by All copyright. reserved. Accepted alba Nymphaea Articlecruziana Victoria feroxEuryale lutea Nuphar Species *t *t Organ stamen petal sepal paracarpel stamen,fertile stamen, external staminode petalinterior petal sepal stamen petal sepal stamen ep epal al

tip connective adaxial abaxial adaxial abaxial adaxial abaxial adaxial abaxial adaxial abaxial petaloid abaxial adaxial abaxial connective adaxial abaxial Surface connective connective adaxial abaxial adaxial abaxial

tetragonal-polygonal, tabular -slightly tabular tetragonal-polygonal, convex tabular Tetragonal, cells conical-papillate domed tetragonal, tetragonal,slightly convex tabular tetragonal, anticlinals slightly wavytetragonal-elongated, convex, tetragonal tabular tetragonal, Epidermis tetragonal to to tetragonalslightlypolygonal, convex slightly tetragonal-elongated, convex slightly tetragonal-elongated, convex slightly tetragonal-elongated, convex to tetragonaltabularpolygonal, ridged papillate,polygonal, ridged papillate,polygonal, ridged papillate,polygonal, ridged papillate,polygonal, ridged papillate,polygonal, domed tetragonal, occasionally , papillate domed, tetragonal, occasionally papillate domed tetragonal, domed tetragonal, domed tetragonal, domed tetragonal, tabular Tetragonal, sparse dense absent sparse sparse dense absent sparse present Hydropotes sparse sparse sparse sparse sparse absent absent absent present present absent sparse sparse sparse sparse sparse sparse absent absent absent absent sparse dense absent dense absent present present present present absent present absent present sparse sparse dense sparse absent absent dense t absen absent present Stomata This article is protected by copyright. All rights reserved. This article isprotected rights by All copyright. reserved. matrices. Delta scores, calculated using SplitsTree,are included. also Table 2. MATRIX 4 MATRIX 3 MATRIX 2 MATRIX 1 examined.*Information fortepal epidermis in Table 1. lotusNymphaea caerulea Nymphaea giganteaNymphaea Accepted Article Description of the trees obtained in the Maximum parsimony analyses ofdifferent the Summary of the cells types and specialized cells indifferent the organs ofspecies the stamen carpellary tip carpellarytip stames petal sepal stamen petal sepal 1 15 1 9 TREES NUMBER OF

connettive adaxial abaxial adaxial abaxial tip connective adaxial abaxial adaxial abaxial connective

N.lutea 88 82 85 81 LENGTH anticlinals tetragonal-elongatedcells,multipapillate wavy multipapillatefaintly cellpolygonal tabular multipapillate tetragonal-elongated, multipapillatepolygonal, multipapillate tetragonal-elongated, multipapillate tetragonal-elongated, multipapillate tetragonal-elongated, to tetragonaltabularpolygonal, multipapillatepolygonal, polygonalmultipapillate multipapillate anticlinals tetragonal-elongatedcells,multipapillate wavy

from Warnerfrom et al, 2008. 0.7705 0.7500 0.7719 0.7547 CI

0.6818 0.6000 0.6750 0.5938 RI

0.3339 0.3827 0.3196 0.3775  SCORE sparse sparse sparse present absent dense dense dense present sparse absent absent present sparse absent absent present rare rare sparse dense absent absent absent sparse sparse sparse absent This article is protected by copyright. All rights reserved. This article isprotected rights by All copyright. reserved. Character 3: Papillae: 0) Absent; 1) Multiple per cell; cell. Single2) per Character 2: Epidermal cell elongation thein petals: 0) Elongated; 1) Isodiametrical. Character 1: Epidermal differentiation between petals and stamens: Absent 0) or light; Pr 1) Appendix theof different matrices. Table 3. Brachyceras Brachyceras

Accepted Article clade Night-blooming Tropical Victoria Support (posterior probability) for differenthypotheses inBayesian the inferenceanalyses Monophyletic Nymphaeas Nymphaeas + Nymphaea Nymphaea Anecphya

+ Euryale

equal equal Matrix1 0.20 0.20 0.31 0.67 0.39 0.59 gamma Matrix1 0.12 0.12 0.27 0.74 0.31 0.50 equal equal Matrix2 0.51 0.51 0.76 0.52 0.51 0 gamma Matrix2 0.50 0.50 0.85 0.49 0.51 0 equal equal Matrix3 0.10 0.10 0.28 0.70 0.30 0.48 gamma Matrix3 0.21 0.21 0.81 0.27 0.45 0.9 0.9

equal Matrix4 0.44 0.44 0.81 0.47 0.12

esent. esent. 0 gamma Matrix4 0.42 0.42 0.88 0.45 0.48 0