Vegetative andreproductiveinnovations of earlylandplants: implicationsfor a uniŽedphylogeny

KarenSue Renzaglia 1, R. Joel Du¡1{,DanielL. Nickrent 1 and David J.Garbary 2 1Department of PlantBiology and Center for Systematic Biology,Southern Illinois University,Carbondale, IL6 2901-6509,USA (renzaglia@plant.siu.edu ) 2Department of Biology,StFrancis Xavier University,Antigonish, Nova Scotia,Canada B2G 2W5

Asthe oldest extantlineages of land plants, bryophytes provide a livinglaboratory in which to evaluate morphologicaladaptations associated with early land existence. Inthis paperwe examine reproductive and structural innovationsin the gametophyteand sporophyte generations of hornworts, liverworts, mosses andbasal pteridophytes. Reproductive features relatingto spermatogenesis andthe architecture ofmotile malegametes areoverviewed and evaluated from an evolutionaryperspective. Phylogeneticanalyses of a dataset derivedfrom spermatogenesis andone derived from comprehensive morphogenetic data are comparedwith a molecularanalysis of nuclearand mitochondrialsmall subunitrDNA sequences. Althoughrelatively small becauseof arelianceon waterfor sexual reproduction, gametophytes of bryo- phytesare the most elaborateof those producedby any land plant. Phenotypic variability in gametophytichabit ranges from leafy to thalloid forms withthe greatest diversityexhibited by hepatics. Appendages,including leaves, slime papillaeand hairs, predominate in liverworts and mosses, while hornwortgametophytes are strictly thalloidwith no organized external structures. Internalizationof reproductiveand vegetative structures withinmucilage-¢ lled spaces is anadaptive strategy exhibited by hornworts.The formative stages ofgametangial development are similar inthe three bryophytegroups, withthe exceptionthat inmosses apicalgrowth is intercalatedinto early organogenesis, a featureechoed inmoss sporophyteontogeny . Amonosporangiate,unbranched sporophyte typi¢ es bryophytes,but developmentaland structural innovationssuggest the three bryophytegroups diverged prior to elaboration of this generation.Sporo - phytemorphogenesis in hornworts involves non-synchronized sporogenesis and the continuedelongation ofthe singlesporangium, features uniqueamong archegoniates. In hepatics, elongation of the sporophyte seta andarchegoniophore is rapidand requires instantaneouswall expandability and hydrostatic support. Unicellular,spiralledelaters andcapsule dehiscence throughthe formationof four regular valves are autapomorphiesof liverworts. Sporophytic sophistications in the moss cladeinclude conducting tissue, stomata,an assimilative layer and an elaborate peristome forextended spore dispersal. Characterssuch asstomata and conducting cells that areshared among sporophytes of mosses, hornwortsand pterido - phytesare interpreted asparallelismsand not homologies. Our phylogeneticanalysis of three di¡erent datasets isthe most comprehensiveto date and points to a singlephylogenetic solution for the evolutionof basal embryophytes. Hornworts are supported as the earliest divergentembryophyte clade with a moss/liverwortclade sister totracheophytes. Among pterido - phytes,lycophytes are monophyletic and an assemblage containing ferns, Equisetum andpsilophytes is sister toseed plants.Congruence between morphological and molecular hypotheses indicates that these datasets aretracking the same phylogeneticsignal and reinforces ourphylogenetic conclusions. I t appearsthat totalevidence approaches are valuable in resolving ancient radiations such asthose charac- terizingthe evolutionof earlyembryophytes. More informationon landplant phylogeny can be foundat: http://www.science.siu.edu/landplants/index.html. Keywords: bryophytes;embryophytes; gametophyte ;landplant phylogeny ;morphogenesis; spermatogenesis

that werefuelled by stochastic geneticchanges and culled 1.INTRODUCTION bynatural selection (Niklas1 997;Bateman et al. 1998). Theearly evolution of land plants was characterized by Poisedat the extremity ofan uninhabited landscape, highrates ofmorphological and reproductive innovations transitionalstreptophytes (greenplants) experienced a burst ofdiversi¢ cation that resulted inradiation into and {Present address:Department of Biology ,The Universityof Akron, Akron,OH 44325-3908, USA. exploitationof avarietyof new terrestrial sites. Repeated

Phil. Trans.R. Soc.Lond. B (2000) 355, 769^793 769 © 2000The RoyalSociety 770K. S.Renzagliaand others Vegetative and reproductive innovations ofearly land plants andsimultaneous patterns ofextensive morphological wereview and update two previously published morpho- diversi¢cation followed by widespread decimation logicaldata sets (Garbary et al. 1993;Garbary & Renza- presumablycharacterized these earlystages ofland colon- glia1 998)and a moleculardata set that combines ization(Gould 1989; Kenrick & Crane1 997 a). Of the sequences ofboth nuclear and mitochondrial small magnitudeof morphological experiments that were subunit(SSU) rDNA. attempted, onlylimited fragments havepersisted through the millennia,including the three lineagesof bryophytes 2.CELL STRUCTURE (Stewart &Rothwell1993; T aylor& Taylor1 993). Giventhe time since divergence,the depauperatefossil Amongembryophytes, there is remarkableconsistency recordand the vastness ofthe newlyinhabited landscape, inthe ultrastructure ofparenchyma cells. The`typical’ it is notsurprising that the earlyphylogenetic history of livingphotosynthetic cell containsa largecentral vacuole, landplants remains oneof the majorunresolved problems aperipheralnucleus, mitochondria, endoplasmic reti- inevolutionary biology .Axiomaticto current evolu- culumand lenticular chloroplasts equipped with grana tionarythought is the conceptthat embryophytesare andscattered starch (Gunning& Steer 1996).Thebryo- monophyletic,that bryophytesrepresent the basalclades phytesand lycophytes exhibit the greatest diversityin andthat Charophyceae,especially Coleochaetales and organellarcomplement and¢ nestructure, andthese Charales,are the closest livingalgal relatives of arche- cellularfeatures provideimportant clues asto evolu- goniates(McCourt 1995).Beyondthese acceptedmaxims tionaryrelationships (Duckett &Renzaglia1 988;Brown there isnoconsensus astointerrelationships amongbryo- &Lemmon 1993).Solitaryplastids (monoplastidy)are phytesand basal pteridophytes. Indeed, virtually every foundin most vegetativecells ofhornworts and charac- conceivablehypothesis regarding bryophyte phylogeny terize mitotic cells ofrepresentative taxaand/ ortissues hasbeen proposed: from paraphyletic in a varietyof (especiallyspermatogenous tissue) ofall bryophytes and branchingorders, tohornworts basal and mosses and lycophytes(Brown & Lemmon 1990 a,1993).Likewise, liverwortsmonophyletic, to a completelymonophyletic monoplastidicmeiosis (discussed below)is distributed bryophyteassemblage (Bopp & Capesius1 996;Capesius amongrepresentatives ofall these clades.Based on the &Bopp1 997;Bremer et al.1987;Garbary et al. 1993; widespreadoccurrence ofmonoplastidy in charophycean Garbary& Renzaglia1 998;Hedderson et al. 1998; Lewis algaeand the exclusivedistribution of this conditionamong et al. 1997;Kenrick & Crane1997 a; Malek et al. 1996; basalembryophytes, it is safeto conclude that monoplastidy Mishler &Churchill1 984,1 985;Mishler et al. 1994; Du¡ is plesiomorphic(Brown & Lemmon 1990 a; Graham &Nickrent 1999;Nishiyama & Kato1 999).Clearly,new 1993).Toensure distributionof a plastidinto daughter dataor perhaps new insights onanalysing existing data cells, the divisioncycle of plastids must betightly linked arerequired toresolve this phylogeneticdilemma. tonuclear division in monoplastidic cell lineages.Plastids Bynature of their antiquity,liverworts,hornworts, areintimately associated with microtubule-organizing mosses andbasal pteridophytes represent relics ofa once centres (MTOCs) andthe twoform the focalpoints for more diverse£ ora,and, as the oldest livingremnants of productionof spindlemicrotubules. earlyland colonization, these organismsprovide a living Inliverworts, only spermatogenous cells aremonoplas- laboratoryin which to examine early morphological tidic andvegetative cells arepolyplastidic. During the adaptationsto existence onland. In this paper,we mitotic cycle,well-de¢ ned, electron-dense aggregations, exploremorphological and developmental features ofbryo- the so-calledpolar organizers, organize the spindles. phytesand basal pteridophytes at the cell,tissue, organ Thesestructures alsocharacterize the monoplastidic andwhole organism levels. Whenever possible,evidence is dividingcells inspermatogenous tissue ofliverworts. presented frommore informativebasal representatives of Endoplasmicreticulum withinthe polarorganizer is the majorclades. This comparative approach is designed connectedto the nuclearenvelope and in this regard toidentify successful phenotypicinnovations of both the resembles the nuclearenvelope-based centrosome of gametophyteand sporophyte, which enabled these plants mosses andtracheophytes (V aughn& Harper 1998).Late tooptimize vegetativeand reproductive activities ina spermatogenouscells ofall archegoniates have centriolar terrestrial setting. Inaddition to illuminating phyloge- centrosomes andthis enablesdirect comparisonwith netic a¤nities, contemplationof morphological and similar algalMTOCs (see below). morphogeneticstrategies providesvaluable insight into Examinationof the internalstructure ofhornwortplas- the directionand sequence ofcharactertransformation. tids providesevidence of further retention ofalgal Thisreview is notintended to be a comprehensive features. Pyrenoidsare found in most anthocerotetaxa overviewof morphology in bryophytes but rather the goal and,as in algae, these electron-dense intrachloroplast istoexplore answers to the followingquestions relatedto bodiesare the site oflocalization of R UBISCO(V aughn evolutionaryadaptations exhibited by early plant life on et al. 1990).Granaof anthocerotes lack end membranes land.What arethe vegetativeand reproductive innova- typicalof other embryophytesand, as in Coleochaete , thyla- tions that enabledthe three bryophytelineages to persist ? koidstraverse anddissect the pyrenoidinto subunits Howdo these organismscope with the constraints of (Vaughn et al. 1992).Channelthylakoids, found in algae terrestrial existence, especiallydesiccation and gravity ? butnot other land plants, further pointto these organelles And¢ nally,whatcan be gleaned from careful scrutiny of asprimitive instructure. Physiologicalspecializations extantbasal embryophytes about morphological char- apparentlyare associated with this chloroplastmicro- acter evolution,including divergences, convergences, anatomy,especiallymechanisms concentratingCO 2, and parallelismsand homologies ?Thesequestions providethe these mayhave been bene¢ cial in a landhabitat (Smith contextfor comprehensive phylogenetic analyses in which &Gri¤ths 1986 a,b).

Phil.Trans.R. Soc.Lond. B (2000) Vegetative and reproductive innovations of early land plants K.S.Renzaglia and others 771

Althoughchloroplast structure inhepatics is similar to generararely grow upright; interestingly , Hymenophyton that ofmosses andtracheophytes, liverworts possess a and Symphyogyna ,twonotable exceptions in the simple uniqueand diagnostic single membrane-bound oil body . thalloidhepatics, both possess well-developedinternal Oil bodiesare not found in all liverworts but they are water-conductingsystems (Ligrone et al.,this issue).The widespreadin basal taxa, suggesting loss inmore derived erect habitfacilitates exploitation of air for exchange of lineages.Originating in meristematic cells fromendo- gasesand light trapping. I talsoprovides a means for plasmicreticulum (Duckett &Ligrone1 995),the oilbody elevatingthe attachedsporophyte and thus promoting hasno counterpartin any other plant group. The function sporedispersal. At issue forboth the thalloidand leafy ofthese organellesis problematicas they apparently are growthforms is waterconservation and protection of notused asafoodreserve. vulnerablegrowing tissues andreproductive organs. Asingleapical cell is responsiblefor growth of bryo- phytegametophytes. Apical cell geometrymay be one of 3.VEGETATIVE GAMETOPHYTES fourfundamental shapes: wedge-shaped (cuneate) ,lens- Thephrase ` gametophytedominant’ distinguishes bryo- shaped(lenticular) ,tetrahedral(pyramidal) or hemidis- phytesfrom all other embryophytes. With the sexual coid(Crandall-Stotler 1 986;R enzaglia1 982).Pyramidal phaseas the prominentpersistent generation,modi¢ ca- systems aretypically associated with leafy habits, lenti- tions ofthe bryophytegametophyte entailed adaptive cularsystems withhighly £ attenedthalli, often with walksthat optimizedvegetative elaboration while simul- thickenedmidrib andmonostromatic wings (e.g. Metz- taneouslyfacilitating water-dependent sexual reproduc- geria and Pallavicinia ),andwedge-shaped and hemidiscoid tion.In this regard,bryophytes and pteridophytes are apicalcells areresponsible for thalloid growth forms, amphibious,i.e. they require uninterrupted access to includingthose ofcomplex thalloid liverworts and horn- waterfor reproductive success andcontinue with vegeta- worts.In mosses, pyramidalsystems predominatewith tiveactivities duringtimes ofwater deprivation. Bryo- the rare secondaryderivation of a lenticularsystem phytesdi¡ er frompteridophytes in that it is the (Crandall-Stotler1 984).Similarly,the wedge-shaped gametophytealone that contendswith these often- apicalinitial is widespreadin hornworts with a develop- con£icting roles ofsexual reproductive and vegetative mentaland evolutionary transformation to hemidiscoid- persistence anddispersal. In pteridophytes, the sporo- basedgenerative cells in Dendroceros (Renzaglia1978) . phyteis the phaseresponsible for production of biomass Hepatics arethe most diverse inthat allfour apical cell andcolonization of newsites. Itis notsurprising therefore shapesoccur in various taxa and developmental modi¢ ca- that fromperusal of structural complexityin gameto- tions ofcell geometryare commonplace. Documentation phytesamong land plants, two features areevident: (i) exists forontogenetic transformation of cell shapefrom this generationnever attains great stature and(ii) bryo- tetrahedralto lens-shaped, from lens-shaped totetra- phytegametophytes are the most elaborateof the land hedralor wedge-shaped, and from wedge-shaped to plants.Thus, bryophytes provide an opportunity to eval- hemidiscoid(F ulford1 956;Renzaglia 1 982;R enzaglia& uatethe structural strategies that enabledrobust gameto- Bartholomew1985) .Often these geometric transitions are phytesto endure in an environment that presented associatedwith a changein gross morphology and/ or extreme selective pressures fora motility-basedfertiliza- orientationof growth. F orexample, the transitionfrom tionsystem. Weacknowledgethat physiologicalmechan- juvenileto mature habitis accompaniedby a transforma- isms forcontending with life in the air,including tionfrom lenticular to tetrahedral apical cell geometryin elaboratebiochemical pathways for desiccation tolerance certain leafyhepatics (F ulford1 956).In Fossombronia , a andthermotolerance in many mosses andpteridophytes, tetrahedralapical cell forms immediatelyin the upright playeda signi¢cant role in plant evolution (Oliver & sporelingand limited segmentationproduces three rows Wood1 997;Kenrick& Crane1997 a; Hanson et al. 1999). ofleaves (Renzaglia & Bartholomew1 985).Further However,it is notwithin the scopeof this reviewto obliquedivisions convert the tetrahedralapical cell toa discuss these physiologicalstrategies. lenticularcell andthis changeresults inthe mature Twofundamental growth forms areexhibited by bryo- thallushabit with two lateral leaf rows. Concomitant phytes:a £attenedprostrate thallus(hornworts and withthis metamorphosis is aswitch inorientation of complexthalloid and simple thalloidhepatics) andan growthfrom vertical to horizontal. The discussion above erect orcreepingcylindrical leafy shoot (leafy liverworts, serves toillustrate that:(i) conversionof apical cell from selected simple thalloidliverworts and mosses) .These onegeometric shapeto another is acornerstone of growthhabits correspond to the optimaloblate spheroid normaldevelopmental processes inbryophytes and is andcylindrical life forms hypothesizedfor small multi- especiallywidespread in liverworts; (ii) changesin apical cellularterrestrial organismsin morphospace (Niklas cell shapeare accompanied by changes in both growth 1997).Comparedwith spherical growth forms that are formand thallus/ shootorientation ;and(iii) becauseof foundin representative greenalgae, dorsiventrally the `ease’ withwhich apical cell shapeis modi¢ed and the compressed thallireduce totalsurface areawhile profoundin£ uence this modi¢cation has on habit, this providingmaximum surface area to volume for gas process alonemay well have provided signi¢ cant exchangeand light harvesting (Niklas 1997) .Leafyforms morphologicalvariation for major evolutionary change. segregatevegetative functions into specialized organs; Crandall-Stotler( 1980,1 984,1 986)clearly documented £attened leavesmaximize light capture and enhance afundamentaldi¡ erence betweenmost mosses andleafy photosyntheticcapacity while the central cylindricalstem liverworts( Jungermanniales)in segmentation of the enhanceswater conservation and enables exploratory pyramidalapical cell andsubsequent leafdevelopment. growth,including upright growth in some taxa.Thalloid Spiralled,undivided leaves in mosses versus three rowsof

Phil. Trans.R. Soc.Lond. B (2000) 772K. S.Renzaglia and others Vegetative and reproductive innovations of early land plants bi¢d leavesare the rudiments ofthese twodistinct onto- some mosses andfurther serve toprotect againstdesicca- geneticpatterns. Asconcluded by Crandall-Stotler tionand damage (Crum &Anderson1 981). (1980),these underlyingdevelopmental discrepancies Inmosses, branched¢ lamentousprotonemata develop supportthe conceptof independent origin of leaves in uponspore germination, and unlike sporelings of liver- mosses andliverworts. W efurther suggestthat additional worts andhornworts, these juvenilestages havethe di¡erences indevelopmentaldesign provide evidence that abilityto produce and disperse multiple leafyshoots leavesor leaf-like structures (`leaves’)have evolved at derivedfrom a singlespore. This latter featureis particu- least twice inmosses andperhaps multiple times inliver- larlyadaptive in acrocarps that donot produce creeping worts.Production of the dissected leaves(phyllids) of shootsand rarely branch. Takakia is decidedlydi¡ erent fromleaf development in Theexclusively thalloid hornworts do not produce Sphagnum andbryopsid mosses (Crandall-Stotler1 986), leaves,slime papillae,scales orany organized external suggestingthat leafevolution in Takakia wasindependent appendage.Indeed, these plantsperform virtuallyall ofthat in other mosses. Becauseof di¡ erences inapical vegetativeand reproductive functions in undi¡ erentiated organizationand the phyleticdistance between taxa such parenchymacells orwithin internal thallus chambers as Haplomitrium , Treubia, Fossombronia , Noteroclada , Sphaero- (Renzaglia1 978;R enzaglia& Vaughn20 00).Perhaps carpos and Blasia,it is possiblethat `leaf’acquisitionis oneof the fundamentalfeatures that hasbeen instru- homoplasticin allof these taxa. mentalin the survivalof anthocerotes is the abilityto Leavesnot only function as photosynthetic organs but producecopious mucopolysaccharides (mucilage) in theyalso provide protection for the fragilegrowing tips. virtuallyany cell, apparentlyin response toenviron- Leafprimordia overarch and surround the apicalcell and mental cues. Mucilagesare carbohydrates that are immediate derivatives.Controlled production of stalked concernedwith imbibition and retention ofwater . mucilage-secreting hairs(slime papillae)accompanies Althoughmosses andliverworts produce mucilage, this leafdevelopment in mosses andliverworts and further substance, withrare exceptions (e.g. the ventralsurface of a¡ords protection against damage and desiccation. In the the thallusof Treubia andthe undergroundaxes of Calo- absenceof leaves, abundant uniseriate tobranched slime bryales),is producedonly in slime papillae.In hornworts, papillaesurround and protect the meristematic regionof protectionof the growingnotch is a¡orded by mucilage simple thalloidliverworts (Renzaglia 1 982;Duckett et al. secretion fromepidermal cells ofyoungapical derivatives. 1990).Inaddition to slime papillae,£ attenedmulticel- Scattered internalcells containmucilage in most taxa lularscales withmarginal papillae provide an alternative andlarge mucilage-¢ lled chambers arecommon in Antho- butequally e¡ ective protectivestructure in Blasia, Cavicu- ceros andselected species ofothergenera. During develop- laria andcomplex thalloid liverworts. ment, antheridialchambers containmucilage and Incontrast tomosses, liverwortevolution has involved mucilagesurrounds the exteriorof archegonial necks. selection towardsdorsiventrality .Schuster (1979,1 984 a,b) Capitalizationon mucilage production and the abilityto suggestedthat the ancestralliverwort gametophyte was a sequester this hydrophilicsubstance ininternal chambers branched,lea£ ess, uprightaxis, much likethat of most areintegral processes that bestowedpartial tolerance to Devoniangametophytes, but lacking conducting tissue dryingconditions in these small thalloidtaxa. andstomata (Remy et al. 1993).Inextant hepatics, erect, Afurther evolutionaryinnovation that almostcertainly radiallysymmetrical shootswith three equalrows of contributedto the vegetativeand reproductive success of leavesare considered plesiomorphic, while procumbent hornworts(as measured bylength of time inexistence) thalloidor leafyforms withreduced to absent underleaves wasthe abilityto form schizogenous spaces. Essential to areviewed as specialized. Appression to the substrate this process is the separationof adjacent cells atthe maximizes contactwith water sources andthus minimizes middlelamella followed by controlled division and expan- potentialdamage associated with desiccation. In leafy sionof cells arounda chamberor canal. In addition to liverworts,prostrate habitshave evolved as anadaptation exclusivelymucilage-containing cavities, hornworts tohorizontal and vertical substrates. Ingeneral, incubous sequester antheridiaand Nostoc coloniesin such chambers. leafinsertions areassociated with vertical substrates (e.g. Thismotif ofinternalization coupled with cell separa- tree trunks) whilesuccubous phenotypes predominate on tionand mucilage proliferation is embodiedin the horizontalsubstrates. developmentof Nostoc coloniesin anthocerotes (R enzaglia While erect radialhabits are exhibited by relatively 1978).Thissymbiotic relationship between the hornwort fewbasal taxa of hepatics, isophyllous acrocarps anda nitrogen-¢xing bacterium hasenabled these plants comprise approximatelyhalf of all mosses (Crum & to£ ourishunder conditions of lownitrogen. The develop- Anderson1981 ).Evolutionaryadaptations that have ment of Nostoc coloniesis ubiquitousin anthocerotes and enabledmosses togrow upright and persist inrelatively beginsnear the apicalcell withthe apparentlyrandom dryenvironments include the acquisitionof more exten- separationbetween two adjacent epidermal cells inthe siveand specialized conducting tissues (He¨bant1 977; ventralthallus. The resulting mucilagecleft providesa Ligrone et al.,this issue),the thickeningof cell wallsand portthrough which soil-dwelling cyanobacteria may accumulationof polyphenolic compounds in these walls, enter the thallus.With maturationof the thallus,a schizo- andthe exploitationof metabolic pathways that confer genousmucilage-¢ lled chamber forms internalto the cleft desiccationand thermal tolerance.Moreover, growth of andexpands with multiplication of cells ofthe Nostoc multiple individualsin dense mats andtufts e¡ectively endosymbiont.Thallus cells elongateand grow among the facilitatesabsorption and retention ofwater in many cyanobacterium.The mature colonymay be rather exten- mosses. Abundantuniseriate, branchedto scale-like para- siveand often forms amoundthat projects fromthe phylliaand pseudoparaphyllia are found on branches of ventralthallus.

Phil.Trans.R. Soc.Lond. B (2000) Vegetative and reproductive innovations ofearly land plants K. S.Renzaglia and others 773

Figure1. Scanning electron micrographs of cultured pteridophyte gametophytes provided by Dean P. Whittier.( a) Young gametophyteof Huperzialucidula .Meristematiczone (M) inagroovethat continues around laterally. Rhizoids (R) originate fromventral derivatives, while uniseriatehairs and gametangia (not produced yet) are formed on adorsalcrown. Bar 0.1 mm. ˆ Micrographby Angel R. Maden.( b,c) Tmesipterislanceolata . (b)Cylindricalgametophyte with abundantuniseriate hairs (H), archegonia(A) andrhizoids (R). The growingtip appears to beunprotected. Bar 0.1 mm. (c)Enlargedview of thegrowing tip ˆ showingtriangular pyramidal apical cell (AC) surroundedby packetsof cells.The prominentwalls that outline cell packets (merophytes)represent original segmentation from the apical cell. Bar 0.05 mm. ˆ

Inhepatics, Nostoc coloniesare restricted totwo sister Whittier, unpublisheddata) are non-photosynthetic when genera, Blasia and Cavicularia (Renzaglia1 982).Acursory youngand convert to green forms whenexposed to light. comparisonof Nostoc colonyproduction in Blasia with Epiterrestrial gametophytesof Palhinhaea, Lycopodiella , that inanthocerotes exempli¢ es highlydivergent develop- Pseudolycopodiella and Equisetum typicallysurvive for a single mentalstrategies forattaining functionally similar struc- growingseason but in some instances maybear several tures inthe twobryophyte groups. In Blasia, Nostoc is sporophytes(Bierhorst 1971;Duckett &Duckett 1980). housedin external ` organs’on the ventralthallus. This so- Apicalgrowth is accomplishedby a meristematic zonein calledauricle is producedin a controlledfashion from Equisetum (Duckett 1970)and in lycophytes (¢ gure 1 a) but apicalderivatives and originates as a mucilagehair, involvesa well-de¢ned tetrahedral apical cell in Psilotum whichundergoes extensive elaboration. Nostoc enters the and Tmesipteris (¢gure 1b,c).Protectionof the growing auriclewhen it is asmall,dome-shaped structure, and regionand gametangia is accomplishedby production of concomitantgrowth of Nostoc andthallus intermixes cells glandularand non-glandular hairs, rhizoids and limited ofthe plantand prokaryote. Continued auricle expansion mucilageproduction (Whittier &Peterson 1984).Often results ina massive externalstructure that super¢cially apicesof subterranean gametophytes are not surrounded resembles the Nostoc colonyof hornworts. byprotective structures (¢gure 1 b,c).Althoughno leaves Havingconsidered apical growth, protective structures areproduced by any tracheophyte gametophyte, the capa- andhabit variability in the three bryophytegroups, cityto form abundant hairs and papillae is reminiscent of comparisonswith basal pteridophytes are in order .In mosses andliverworts. However, conducting tissue, which general,gametophytes of pteridophytesare not as compli- is commonplacein mosses andmore restricted inoccur- catedor long-livedas those ofbryophytes(Bierhorst 1971; rence inliverworts,is adevelopmentalanomaly in gameto- Gi¡ord & Foster 1988;Bell 1994; Whittier 1977,1981, phytesof extant pteridophytes (Bierhorst 1971). 1983;Whittier &Webster 1986;Whittier &Thomas Fungalassociations are widespread in gametophytes of 1993).Althoughvariability exists amongthe genera,two pteridophytes,liverworts and hornworts, but are lacking fundamentalgrowth forms predominatein basal tracheo- inmosses (Duckett et al.1991).Fungalhyphae facilitate phytes:(i) greenepiterrestrial forms withirregular absorptionand translocation of minerals, waterand uprightlamellae and (ii) thick,£ eshysubterranean forms organicmolecules withinthe plant.In nature, hetero- witha fungalsymbiont. The latter type,exempli¢ ed by trophicsubterranean gametophytes of pteridophytes rely Huperzia (¢gure 1a), Lycopodium, Diphasiastrum , Phegmar- exclusivelyon endophytic fungi for nutrient uptake. iurus,Psilophytes(¢ gure 1 b), Botrychium and Ophioglossum , Fungal^plantsymbioses wereprobably established inthe is generallyconsidered ancestral. These gametophytes primitive archegoniatesand have developed repeatedly maypersist forseveral years and they have the capacity duringthe evolutionof land plants (Pirozynski & toproduce multiple sporophytesand form abundant Malloch1975; Pirozynski 1 981).Fora discussionof regenerants.The gametophytes of Phylloglossum (Whittier symbioticinteractions among fungi and bryophytes see &Braggins1992) and Huperzia (A.R. Madenand D .P. Read et al. (this issue).

Phil. Trans.R. Soc.Lond. B (2000) 774K. S.Renzaglia and others Vegetative and reproductive innovations of early land plants

hornworts (a) (b) (c) (d) (e)

mature neck

neck neck canal cells venter ventral canal cell egg pteridophytes (i) (j) (f) (g) (h)

neck

neck canal cells mature neck ventral canal cell egg

Figure2. Comparison of archegonialdevelopment in hornworts( a^e)andpteridophytes ( f^j ). (a)Longitudinalview and (b)cross-sectionalviewof archegonialinitial traversed by threeanticlinal walls that delimit a centralaxial cell and three peripheralcells. The axialcell gives rise to theneck canal cells, ventral canal cell and egg while theperipheral cells form sterile outerlayers. ( c)As seenin cross-section,three additional segments in theperipheral cells form six rows of neckcells. ( d ) Light micrographsurface view of sixrows of neckcells in Phaeoceroslaevis . (e)Longitudinalsection of thesunken archegonium of Phaeoceroslaevis . ( f )Longitudinalview and ( g)cross-sectionalviewof archegonialinitial traversed by singlepericlinal wall that delimitsan outercell that forms the neck and an innercell that gives rise to neck canal cells, ventral canal cell and egg. ( h) As seenin cross-section,successive anticlinal divisions in theouter cell form four rows of neckcells. ( i)Scanningelectron micrograph ofarchegonialneck with fourrows of cellsin Psilotumnudum . ( j )Longitudinalsection of sunkenarchegonium of Pteridium aquilinum.

4.REPRODUCTIVE GAMETOPHYTES Selectivepressures ina terrestrial environmentfor reproductiveprocesses that require uninterrupted access towater are indeed extreme. Thetransmigration of algae toland and the subsequent evolutionof embryophytes necessitated the evolutionof multicellular sex organs (Niklas1997) .Theseorgans not only served toprotect vulnerabledeveloping gametes butalso the originof multicellularfemale sex organs (archegonia) was a requi- site forembryo development, a universalfeature of land plants.The evolution of gametangia in archegoniates undoubtedlywas a complexhistorical process that required coordinationof morphogenetic with physio- logicaland ecological signals. F orexample, production of sexorgans must betimed appropriatelyand must occur rapidlyin response to£ uctuatingseasonal conditions. Gametangiamust bestrategicallyplaced on the plantand protectivestructures amplydeveloped. In gametophyte- dominant,land-dwelling plants the compromise between optimizationof vegetative growth and the constraints of Figure3. Light micrograph of antheridiumof Takakia ceratophylla .Longitudinalsection of anteriorof developing, sexualreproduction often resulted inthe abandonmentor elongatedantheridium. Cells are in packetsarranged in two reducedemphasis ongeneticexchange (i.e. elaborationof rowsthat represent the original biseriate ¢ lamentproduced mechanisms forasexual reproduction and self-fertilization) . byleft^rightdivisions from an apicalcell. The outlineof the Suchstrategies arealso common in algal groups but apicalcell is visible as atriangularwall containing developing evolvedin response todi¡ erent selective pressures. spermcells at theantheridial tip. Bar 10.0 m m. ˆ Inan attempt tounderstand adaptive strategies and characterevolution in the sexualphase of early arche- Unlikethose ofpteridophytes, gametangia of mosses goniates,we will brie£ y examineand compare: (i) sex andliverworts are stalked and extend from the epidermal organontogeny and (ii) spermatogenesis amongbasal surface(Bold et al.1987).Inleafy taxa, gametangia are bryophytesand seedless tracheophytes. either associatedwith leaves or leavessurround a terminal

Phil.Trans.R. Soc.Lond. B (2000) Vegetative and reproductive innovations ofearly land plants K. S.Renzaglia and others 775

(b) hornworts

(a)

chamber roof schizogenous space antheridial initials

antheridial complex

Anthoceros pteridophytes (d)

(c)

jacket

spermatogenous tissue

antheridium

Ophioglossum

Figure4. Comparison of antheridialdevelopment in hornworts( a,b)andpteridophytes ( c,d). (a)Inhornworts,a periclinal divisionin asuper¢cial cell delimits a subepidermalcell that gives rise to upto25antheridiaand an epidermalcell that produces achamberroof two cells thick. A schizogenousspace forms the cavity that houses the antheridia. Antheridial development from initialsat thebase of thecavity follows a developmentalpathway similar to otherbryophytes. ( b)Lightmicrograph of antheridial complex in Anthocerosagrestis . (c)Inpteridophytes,a periclinaldivision in asuper¢cial cell delimits a subepidermalcell that gives riseto thespermatogenou stissueand an epidermalcell that forms the sterile jacket. ( d)The singleantheridium that develops fromthis process in Ophioglossumengelmannii . cluster ofsex organs. Abundant papillae, hairs and para- froman apical cell. Inmosses andliverworts, the arche- physesare intermixed amonggametangia and provide gonialinitial elongates and divides above the epidermal further protection.Thalloid liverworts have evolved a surface,while in hornworts, the developingarchegonium vastarray of structures andmechanisms forprotecting remains surroundedby thallus tissue. Thismosaic of gametangia,including inrolled ventral branches ( Metz- characters relatedto archegonial ontogeny provides geria),secondarilysunken chambers ( Pellia, Noteroclada and evidenceof divergent developmental pathways in the complexthalloid liverworts) and lamellar outgrowths of bryophyteclades. However ,whenthe formativedivision the thallus(R enzaglia1 982).Incontrast, gametangiain sequences that establish the archegoniumproper are hornwortsare produced and maintained within the examined,a fundamentalpattern emerges that is diag- con¢nes ofthe thallusand as such super¢cially resemble nostic ofbryophytes (Schuster 1966).Inall three groups, those ofpteridophytes. Gametangia of basal pterido- the process involvesthree longitudinaldivisions that form phytesare never stalked and are either entirelysunken acentraltriangular axial cell surroundedby three withinthe thallusor formconspicuous epidermal mounds peripheralcells (¢gure 2 a,b).Theperipheral cells form (Bierhorst 1971). the neckand venter whilethe axialcell givesrise tothe Toevaluatehomology of gametangia, it is critical to neckcanal cells, ventralcanal cell andegg. F urther divi- examinecell divisionpatterns andespecially the initial sions inthe peripheralcells typicallyresult ina neckof formativedivision sequences duringwhich the develop- ¢veor six cell rows(¢ gure 2 c,d)(Renzaglia1982) . mentalfate of subsequent cell lineagesis determined Thisdivision pattern ismarkedlydi¡ erent fromthat of (Roux1895; Niklas 1 997).Theseprimary divisions are pteridophytes,in which the singleepidermal initial ¢ rst geneticallycontrolled and provide the blueprintfor organo- dividestransversely (periclinal division) .Theouter cell genesis. At¢ rst glance,there appearsto be no similarity then further dividesto form the neckwhile the innercell inarchegonial ontogeny among liverworts, hornworts and dividespericlinally to produce neck canal cells, ventral mosses. Archegonialdevelopment in mosses uniquely canalcell andegg (¢ gure 2 f,g, j)(Bierhorst 1971;Kenrick involvesthe initialproduction of an apical cell that & Crane 1997a).With expansion,the archegonium segments left andright forming a biseriate ¢lament projects fromthe epidermalsurface. The neck invariably (Scho¢eld 1 985;Smith 1955).Thearchegonium proper consists offour vertical rows of cells (¢gure 2 h,i). Clearly, forms fromthe terminal cell inthe ¢lamentafter the there areno parallels in developmentbetween the sunken apicalcell ceases its patterned mitotic divisions.Hornwort archegoniaof hornworts (¢ gure 2 e)andsimilar andliverwort archegonia develop without elongation embeddedarchegonia in pteridophytes (¢ gure 2 j).

Phil. Trans.R. Soc.Lond. B (2000) 776K. S.Renzagliaand others Vegetative and reproductive innovations ofearly land plants

Bryophyteantheridia contain well-developed stalks andglobose to elongated antheridial bodies. As inarche- gonialdevelopment, antheridial ontogeny in mosses is peculiarin that anapical cell is responsiblefor early elongation(Smith 1955).Thisfeature alone, i.e. antheri- dialdevelopment involving an apical cell, enables the identi¢cation of Takakia asa moss (¢gure 3) (Smith & Davison1 993).Thegenesis ofhornwort antheridia within aninternal thallus chamber has been cited asa funda- mentaldeparture in morphogenetic design from all other archegoniates(Crandall-Stotler 1 980;Bold et al. 1987). However,with closer scrutiny,asingleunderlying pattern ofdevelopment is manifest inantheridial development of mosses, hornwortsand liverworts. In all three groups,the antheridialinitial elongates and similar divisioncycles formeither twoprimary spermatogones with four surroundingjacket initials (mosses andleafy/ simple thal- loidliverworts) or fourprimary spermatogones with eight peripheraljacket initials (hornworts and complex thalloid liverworts).Thesesimilarities must represent plesio- morphies ofthe landplant clade. The di¡ erence inhorn- wortsis that the antheridialinitial is locatedat the base ofthe schizogenousantheridial chamber and not at the thallussurface asin other bryophytes.Apparently ,in hornworts,an evolutionary shift indevelopmental poten- tialhas occurred from epidermal (layer surrounding the externalsurface) toepithelial (layer surrounding an internalspace) cells, bothof which are surface cells that enclosetissue. Thedesignation of hornwort antheridia as endogenousrefers onlyto the locationof development Figure5. Longitudinal section of Phylloglossumdrummondii andnot to an inherently di¡ erent developmental bisexualgametangium. In theregion of themature pathway. gametophytewhere antheridial production shifts to Aninitial periclinal division in a super¢cial cell in archegonialproduction, bisexual gametangia are common. hornwortsmimics earlyantheridial development in pteri- This gametangiumhas developing sperm cells (SC) in the dophytes(cf. ¢gure4 a,c).However,unlike in pterido- `neckregion’ that overlie a matureegg cell (E). Bar 20 m m. ˆ phytes,where this initialdivision gives rise toa single antheridium(see below),inhornworts extensive cell divisionsin the resulting twocells producean entire (Renzaglia& Duckett 1988,1991 ;Garbary et al. 1993). antheridialcomplex consisting of a sunkenchamber with Duringspermatogenesis, undi¡erentiated parenchyma overlyingtwo -layeredroof and up to 25 enclosed anther- cells areprogressively transformed intostreamlined, idia(¢ gure 4 a,b). coiledcells containingminimal organelles. The process Antheridialdevelopment is essentiallyidentical to involvesthe originand development of an elaborateloco- archegonialdevelopment in pteridophytes (¢ gure 4 c) motoryapparatus, a structure that enablesunparalleled (Bierhorst 1971).Theprocess beginswith a transverse comparisonswith motile algalcells. Aunifyingfeature of divisionin an epidermalcell. The outer cell givesrise toa motile gametes ofarchegoniates is the de novo origin of sterile jacketand the innercell forms the spermatogenous centrioles inlate spermatogenous tissue (Vaughn& tissue. Sosimilar arethese divisionpatterns inthe two Harper 1998;V aughn& Renzaglia1 998).Inplants with sexorgans of pteridophytes that it is virtuallyimpossible bi£agellated sperm cells, centrioles originateas bicen- todi¡ erentiate antheridiafrom archegonia in earlystages trioles, structures that arecomposed of two centrioles oforganogenesis. Moreover ,inprotandrous lycophytes, attachedend to end. The bicentriole is the landplant whenthe transitionbetween male and female sex organs analogueof the orthogonalcentriolar pair that istypical occurs, it is possibleto ¢ ndbisexual gametangia that ofprotists andanimals. In multi£ agellated gametes, containdeveloping sperm inthe `neckregion’ and an egg centrioles originateas more complicatedspherical orga- cell atthe base(¢ gure 5) (K. S.R enzagliaand D .P. nelles knownas blepharoplasts.In addition to origin and Whittier,unpublisheddata) . developmentof the locomotoryapparatus, histogenesis of malesex organs provides comparative data on cyto- plasmicphenomena such asplastid behaviour and 5.SPERMATOGENESIS cellularpolarity .Numbers oforganelles and their spatial Sperm cells ofarchegoniates are propitiously arrangementsare established duringthe proliferative constructed foroptimal swimming e¤ ciency in a terres- divisionsof organogenesis and the nascent spermatid is trialenvironment. As the most complicatedcells organizedfor expeditious di¡ erentiation into a motile cell producedby archegoniates, motile sperm cells providea (Renzaglia& Duckett 1987;Bernhard& Renzaglia1 995; wealthof developmental and phylogenetic data Renzaglia et al.1994).Theprocess ofspermatogenesis and

Phil.Trans.R. Soc.Lond. B (2000) Vegetative andreproductive innovations of early land plants K. S.R enzagliaand others 777

Figure6. Reconstruction sofmaturebi£ agellated spermatozoids of Charalesand bryophytes. Note sinistral coiling of allbut Phaeoceros (b),which exhibitsa right-handedcoil. Colour coding: red (pink) £agellaand basal bodies; blue nucleus; ˆ ˆ brown mitochondria;yellow splinemicrotubules; orange lamellarstrip; green plastid. (a) Charavulgaris .Adaptedfrom ˆ ˆ ˆ ˆ Duncan et al. (1997). (b) Phaeoceroslaevis. Adaptedfrom Carothers & Duckett(1980) and Renzaglia & Duckett(1989). ( c) Blasia pusilla. Adaptedfrom Renzaglia & Duckett(1987). ( d) Aulacomniumpalustre. Dataderived from Bernhard & Renzaglia(1985). Drawingby H. DeeGates. its bearingon plant phylogenetics will be reviewed else- apparatusand four organelles: an anterior mitochon- where(R enzaglia& Garbary200 0).Here weconsider drion,a compactedcentral nucleusand a posterior generalfeatures ofthe mature spermatozoid,especially of plastidwith an associated mitochondrion (¢ gure 6 b^d ). bi£agellated cells, andevaluate the selective processes Inaddition to £ agellaand basal bodies, the locomotory that haveacted to bring about these phenotypes. apparatusconsists ofa lamellarstrip anda narrowband Bi£agellated sperm cells areproduced by charophycean ofmicrotubules (the so-calledspline) ,whichextend algae(except Zygnematales),bryophytesand most lyco- aroundthe cell providinga frameworkfor positioning of phytes(¢ gures 6and7) ,whileall other tracheophytes organelles.The lamellar strip is composedof centrin, a withmotile sperm producemulti£ agellated cells (¢gures calcium-bindingcontractile protein, and as such functions 8and9) .Themature bi£agellated cell inbryophytesis a asan MTOC for the splinemicrotubules andin posi- helicalcylinder ,withan anteriorlypositioned locomotory tioningthe £agella(V aughn et al. 1993).Atcellular

Phil. Trans.R. Soc.Lond. B (2000) 778K. S.Renzagliaand others Vegetative and reproductive innovations ofearly land plants

Figure7. Reconstruction sofmaturebi£ agellated spermatozoids of lycophytes.Colour coding: red (pink) £agellaand basal ˆ bodies;blue nucleus;brown mitochondria;yellow splinemicrotubules; purple lamellarstrip; green plastid;grey extra- ˆ ˆ ˆ ˆ ˆ ˆ neouscytoplasm. ( a) Selaginellakraussiana. Adaptedfrom Renzaglia et al. (1998). (b) Lycopodiumobscurum. Adaptedfrom Maden et al. (1996). (c) Palhinhaeacernua. Adaptedfrom Robbins & Carothers(1978). ( d) Lycopodiellalateralis. Adaptedfrom Maden et al. (1997). maturity,the lamellarstrip regresses toa dense rim along gametes of Chara and Nitella,the same basicorganization the leadingedge of the cell (¢gure 6 b^d ). asinbryophytes is exhibited;the primarydi¡ erence is in Amongthe algaloutgroups to land plants, only Char- the number oforganelles.Approximately 30 mitochondria alespossess coiledspermatozoids (¢ gure 6 a). In male arealigned at the cell anteriorand up to six starch-¢ lled

Phil.Trans.R. Soc.Lond. B (2000) Vegetative and reproductive innovations ofearly land plants K.S.R enzagliaand others 779

plastids areassociated with scattered mitochondriaat the cell terminus. Gametes of Chara neverpossess alamellar strip butthose of Nitella and Coleochaete possess this componentof the locomotoryapparatus. In contrast to bryophytes,in which the plates ofthe lamellarstrip are orientedat 45 8 tothe longitudinalaxis of spline micro- tubules, inalgal cells this angleis consistently 90 8 (Graham 1993). Mature spermatozoidsof hornworts, mosses andliver- wortsare immediately distinguished from each other by majorarchitectural di¡ erences. Hornwortsperm cells are extremely small ( ca. 3.0 m mindiameter) andbilaterally symmetrical (¢gure 6 b).Thetwo basal bodies insert the parallel£ agellainto the cell anteriorat approximately the same level.U nlikespermatozoids of all other archegoni- ates, whichare sinistrally coiled, hornwort gametes exhibit adextralcoil. In contrast, the bilateralspermatozoids of liverwortsand mosses areasymmetrical (¢gure 6 c,d). In bothgroups of bryophytes,the twobasal bodies are mark- edlydi¡ erent inlength, structure andinsertion intothe cell.As aconsequence,the £agellaare staggered in their emergence fromthe cell. When viewedfrom the cell ante- rior,the basalbody that ispositionedfurther forwardsis situated onthe rightand the more posterior basalbody is onthe left side ofthe cell. Theanterior basal body in both mosses andliverworts contains dorsal microtubule triplets that extendin frontof the basalbody proper. Similarly ,in bothgroups, the posterior basalbody exhibits a uniqueyet consistent structure withventral microtubule triplet exten- sionsaccounting for most ofits length.The striking commonalitiesin construction of this complexlocomotory apparatus,including basal bodies and £ agella,strongly supportmonophyly of liverwortsand mosses (Renzaglia& Duckett 1991;Garbary et al.1993).Theprimary di¡ erences inmoss comparedwith liverwort sperm cells arethe occurrence ofa straymicrotubule and the positionof the plastidand associated posterior mitochondrionalong the innernuclear surface (¢gure 6 d)andnot at the cell terminus asinhepatics(¢ gure 6 c). Lycophytespermatozoids are structurally diverse, probablymore sothan in any other land plant group (¢gures 7and8) (Maden et al. 1996,1997 ;Renzaglia et al. 1998,1999) .Within Lycopodiaceae,gametes maybe bi- £agellatedand coiled (¢ gure 7 b),multi£agellated and coiled(¢ gure 8 a)orbi£ agellated with a more ovoid outline(¢ gure 7 c,d).Thelamellar strip persists inthe mature gameteand the anglebetween lamellar strip and splineranges from 45 8 to 908 (Maden et al.1996,1997) . Comparedwith bryophyte spermatozoids, those oflycop- Figure8. Transmission electron micrographs of multi- sids containmore cytoplasm,including numerous small £agellatedmale gametes of lycophytes.( a) Phylloglossum mitochondriaat the cell posterior.Plastid number varies drummondii.This spermatozoidcontains approximately 20 fromone to ¢ ve.Plastids are either positionedon the £agella(F) alignedin acoilover the multilayered structure innernuclear surface as in the more coiledgametes (MLS)atthecell anterior. The nucleus(N) isbroad,slightly (¢gure 7b)oraggregated at the cell posteriorin ovoid coiledand contains uncondensed inclusions. Numerous mito- cells (¢gure 7 c,d).Apparentevolutionary trends within chondria(MI) anda plastid(P) with largestarch grains are the familyinclude an increase incell lengthand diameter, positionedat therear of thecell. Bar 1.0 m m. (b) Isoe« tes ˆ anda decrease incoiling(Maden et al. 1997).Flagellaare bolanderi.Approximately20 £agella(F) aredistributed along slightlystaggered in the smaller coiledsperm cells of thelength of thisnarrow and coiled spermatozoid. The nucleus(N) isacompactednarrow cylinder and it overlaps Huperzia and Lycopodium andmore widelyseparated in the with twomitochondria (MI). An inconspicuous,posterior more specializedovoid cells of Palhinhaea and Lycopodiella . plastid(P) lacksstarch. Upon motility, the large central Theseideas are contrary to those ofRobbins& Carothers massof cytoplasm(CM) isshed. Bar 1.0 m m.Unpublished (1978),whospeculated that anovoid cell withwidely ˆ micrographby Gayleen Cochran. spaced£ agellais the primitive conditionin archegoniates.

Phil. Trans.R. Soc.Lond. B (2000) 780K. S.Renzaglia and others Vegetative and reproductive innovations of early land plants

Figure9. Scanning electron micrographs of multi£agellated sperm cells of pteridophytes. Bars 1.0 m m. (a) Equisetumarvense whole ˆ spermatozoidwith approximately54 £agella arrangedin coilsaround the cell anterior. The nucleus(N) isbroadin themiddle andtapers on bothends. A splineof upto 300microtubules overlies the nucleus and providesa frameworkfor the coils. Abundantorganelles extend along the inner nuclearregion (not visible). ( b) Higher magni¢cation of anteriorof Equisetumarvense spermatozoidshowing coils along which £agellaare inserted into the cell and narrow anteriorof nucleus(N). ( c)Spermatozoidsof Psilotumnudum arebroad and coiled with approximately36 £ agellainserted along the anteriorcoil. The spline(S), consistingof up to200microtubules, associates in small bandsthat are visible over the broad nucleus.Unpublished micrograph by ThomasJohnson. ( d)The spermcell of Angiopterisevecta resemblesthat of Equisetum and Psilotum inthatit is coiledwith numerous£ agellainserted along the anteriorcoils, and organelles line the inner nuclearregion. Like sperm cells of lepto- sporangiateferns, this cell is more £ attened andribbon-shaped than that of Equisetum and Psilotum. (e)Highermagni¢ cation of anterior of Angiopterisevecta spermatozoid showingribbon-shaped anterior coils along which the£ agellaare inserted into the cell.

Theonly homosporous lycophyte with multi£ agellated Althoughsperm cells of Isoe« tes havenot been thor- sperm (ca.20£ agella)is Phylloglossum (¢gure 8a): this oughlyexamined ultrastructurally ,preliminaryobserva- conditionin a solemember ofthe Lycopodiaceaemost tionsreveal that these cells arecoiled, highly streamlined certainlyis anautapomorphy (R enzaglia& Whittier andpossess approximately20 £ agella(¢ gure 8 b). Unique 1993;Renzaglia & Maden20 00). amongland plant gametes is the lackof a starch-¢lled Thesimilarity in mature gametestructure between plastidin the mature cell.Commonalities with the Selagi- Selaginella andthe bryophytesis intriguingfrom an evolu- nella spermatozoidand marked structural deviationsfrom tionaryperspective. Likebryophytes, Selaginella produces those ofhomosporous lycopsids suggest this cell isderived small,coiled, bi£ agellated sperm cells withfour orga- fromwithin the heterosporouslycopsid lineage and bears nelles arrangedsimilarly along a microtubularspline nodirect evolutionaryrelationship with the multi- (¢gure 7a).Nolamellarstrip exists inthe mature cell. £agellatespermatozoid of Phylloglossum . However,unlike bryophyte spermatozoids, those of Selagi- Theremaining pteridophytes ( Equisetum, Psilotum and nella possess exceptionallylong anterior mitochondria that ferns) producelarge (up to 18 m mindiameter) coiled, occupymore ofthe cell lengththan the nucleus.Compre- multi£agellated sperm cells withabundant organelles hensivedevelopmental data on Selaginella kraussiana reveal (¢gure 9) .Similarities amongall of these cells are further deviationsfrom bryophyte spermatogenesis as numerousand point to a commonorigin. Most notableis wellas substantial similarities withL ycopodiaceaeand the cell anteriorthat bears acoiledlocomotory apparatus other pteridophytes(R enzaglia et al.1999).Thesestudies withover 34 £ agella(¢ gure 9 b,c,e).Abroad,microtubular reinforcethe notionthat developmentaldata are crucial bandoutlines the cell andthe nucleusextends alongthe inthe determinationof structural homology. mid-spline regionto the cell tip (¢gure 9 a^d ).Details of

Phil.Trans.R. Soc.Lond. B (2000) Vegetative andreproductive innovations ofearly land plants K. S.R enzagliaand others 781 spermatozoidstructure inthese plantswill be presented bryophytesporophytes as less complexthan those ofpter- elsewhere (Renzaglia& Garbary20 00). idophytesis anoversimpli¢ cation. In pteridophytes, in Allspermatozoids of land plants exhibit coiling, either additionto facilitating spore productionand dispersal, externalin cell architecture orinternal in arrangementof the sporophyteis responsiblefor vegetative growth, organelles.Coiling is the direct result ofcellularelongation includingmaximum light harvesting and mechanical associatedwith extension of the singleband of splinemicro- support.In bryophytes, where the gametophyteis the tubules withinthe constraints ofa sphericalcell, i.e. vegetativephase, the solefunction of the sporophyteis to compactionand elongationnecessarily parallel the circular produceand disperse spores. Ine¡ ect, eachbryophyte boundariesof the cell.Thecomplicatedlayout of landplant sporophyteis asolitarysporangium, with or without a sperm cells most certainlyprovides a hydrodynamically stalk,that is designedto make and release spores andas soundarchitecture. Cellularelongation or streamlining such hasundergone virtually no selection forvegetative reduces excess baggageand, perhaps even more impor- growth.I tis notsurprising then that bryophytesporo- tantly,is instrumental inmovement of the spermatozoid phytes,although they are unbranched and lack leaves, throughthe narrowtube ofthe archegonialneck. The producesporangia that arethe most complicatedof any convergencein structure ofarchegonia and the eggappa- producedby land plants. Lines ofspecialization have led ratus ofCharales may explain similarities insperm cell toelaborate spore productionand dispersal mechanisms, shapebetween Charales and basal land plants and may such asthe peristome oftrue mosses, cellularelaters of accountfor the markeddi¡ erences betweenthese cells liverwortsand the highlycomplicated sporangium of andthose ofColeochaetales. hornwortsthat continuesto elongate throughout the Inbryophytes and perhaps some lycophytes,sperm growingseason. N oparallelsof such intricate sporangial cells haveremained relatively small andsimple. Thelow complexityare found in any tracheophyte, for indeed haploidDNA contents ofthese plantsare correlated with these roles havebeen taken over by the vegetativesporo- sperm size andare re£ ected insmall cell size (Renzaglia phytictissues, notthe sporangiumitself. et al. 1995).Amongbryophytes, hornworts have the smal- Thisnew look at sporophyte complexity in bryophytes lest genomesize andthe smallest sperm cell. The maybe extendedfurther inan evaluationof reproductive symmetric placementof £ agellaat the anterioris an potentialin sporophytes of embryophytes. Moss, liver- e¡ective organizationto move an extremely small cell wortand hornwort sporophytes are limited intheir throughwater .With greaterDNA contentand increase in capacityfor spore production because individually they cell size, it wouldfollow that asymmetry andstaggering bearonly a singlesporangium. However, when the of£ agellarinsertion maybe necessary tofacilitate move- totalityof sporophytes that arepotentially produced by a ment. Datafrom sperm cell structure suggestthat horn- singlegametophyte is considered,a verydi¡ erent inter- worts divergedfrom other bryophytes before £ agellar pretationemerges. Duringtheir lifetime andeven within staggeringevolved and that mosses andliverworts asinglegrowing season, the typicalsexual bryophyte acquiredstaggering from a commonancestor with larger gametophytegenerates a multitude ofsporophytes. In spermatozoidsthan those ofthe hornwort ^embryophyte gametophytesof `promiscuous’ bryophytes, it is con- progenitor.If cells aresymmetrical, asinhornworts, the ceivablethat the totalnumber ofspores producedby the directionof coiling a¡ ords no selective advantageand is `population’of sporophytes on a singlegametophyte may free tochange without consequence to swimming perform- exceedthat ofa singlepteridophyte sporophyte during a ance.Once asymmetry wasestablished, asin all other growingseason. Moreover ,unlikethe multiple sporangia landplants, the di¡erent constructions ofthe rightand ofa pteridophytesporophyte (generated by a single left cellularhalves may have had `locked in’ genetic deter- gametophyte),the numeroussporophytes produced by a minationof coiling direction. Based on these ideas,the solitarybryophyte gametophyte are potentially products prototypicspermatozoid of archegoniates was probably a ofindependent fertilizations. Given outcrossing, each minute bi£agellated cell withminimal sinistral coiling. sporophyteis geneticallyunique. In bryophytes, evolu- Observationsof remarkablywell-preserved sperm cells in tionhas maximized both sexual reproduction and the Devoniangametophytes strengthen the speculationthat consequentialproduction of genetically diverse primitive landplants produced small coiledgametes sporangia,while in tracheophytes, vegetative growth is (Remy et al.1993;Duncan et al. 1997). responsiblefor increased development of genetically similar sporangia. Amongtracheophytes, each gametophyte of hetero- 6.SPOROPHYTES sporousforms hasthe potentialto produce only a solitary Liverworts,hornworts and mosses arereadily distin- sporophyte.Basal homosporous tracheophytes, in con- guishedfrom polysporangiate tracheophytes by their trast, oftenhave the capacityto produce several sporo- unbranched,monosporangiate sporophytes that arenutri- phytesand longer-lived subterranean gametophytes may tionallydependent on the persistent, photosynthetic yieldmultiple sporophytes(Bierhorst 1971;A.R.Maden, gametophyte.Given the likelyorigin of the sporophyte unpublisheddata) .Subterraneangametophytes are di¤ - througha delayin meiosis (i.e. antithetic theory),the cult to¢ ndin nature and therefore theyhave been in- lackof elaboration of the sporophytein bryophytes is su¤ciently examined. As a result, the extent of viewedas a plesiomorphyamong embryophytes. Indeed, sporophyteproduction by a singlegametophyte in basal contemporaryevidence, both molecular and morpho- pteridophytesremains unknown.N evertheless, it canbe logical,supports the basalposition of bryophytes in land concludedthat the abilityto generate multiple sporo- plantphylogeny (Graham 1993).However,re-evaluation phytessigni¢ cantly increases reproductivepotential in ofsporophyte structure suggests that the interpretationof landplants with persistent gametophytes.

Phil. Trans.R. Soc.Lond. B (2000) 782K. S.R enzagliaand others Vegetative and reproductive innovations ofearly land plants

Becauseselection forindividual sporophytes of bryo- mosses andliverworts. The endothecium in hornworts phyteshas acted to enhance the manufactureand and Sphagnum forms acolumellaonly and is notresponsible dispersal ofspores, it is notsurprising that anumber of forproducing sporogenous tissue asin other bryophytes. diverse mechanisms forelevating the sporangiumhave Asin gametangial ontogeny ,apicalcell involvementis evolved.T oexposespores tosu¤ cient windcurrents, echoedin the formativephase of embryogenesisin mosses. sporangiamust beraised above the gametophytesurface. Later indevelopment,before di¡ erentiation of the capsule, Someof these strategies forsporangial elevation involve anintercalary meristem typicallyforms andbrings about extensionof gametophytic tissue (archegoniophorein further elongationof the sporophyte(R oth1 969).Inboth Marchantialesand pseudopodium in Sphagnum and mosses andhornworts, photosynthetic capability in the Andreaeales),whilemost involvegrowth of sporophytic sporophyteis partiallyresponsible for continued growth. tissue. Inthose withgametophytic elevation of sporangia, Anassimilative layer extends the lengthof the hornwort the seta remains short andthe sporangiumrelatively sporophyte.In mosses, the photosyntheticregion extends simple, e.g.no conducting tissue ispresent in Sphagnum or alongthe entire capsuleand includes internal specializa- Andreaeales.Elaboration of sporophytic tissue entails tionof aerenchyma.In contrast, the hepaticsporophyte is either the productionof aseta (true mosses, Takakia, leafy containedwithin protective gametophytic cells untilspores andsimple thalloidhepatics) orthe developmentof a havematured. Thus,gametophytic nurturing occurs basalmeristem (hornworts). throughthe lifeof the hepaticsporophyte. Inhepatics, seta extensionis rapidand involves the Takenas a whole,the fundamentaldi¡ erences in extensionof turgid cells. Mechanicalstrength isprovided development,structure, andspore dispersal strategies byhydrostatic pressure againstthe cell wallfrom within suggestdivergence of the three groupsof bryophytes prior the largecentral vacuole. Elongation of the archegonio- tospecialization of mechanisms forelongation and phoreand development of protective structures around continuedgrowth. With this scenario,the prototypic the developingsporophyte also entail rapid wall expand- bryophytesporophyte would be a small mass ofcells with abilityand extension of ephemeral organs, an innovation afertile internalregion, as predicted byGraham ( 1993), that is commonin a varietyof hepatic tissues. The Mishler &Churchill( 1984)and Hemsley (1994).Because strategyin these plantsis for`instantaneous’ and e¡ ective aplacenta,with specialized cells atthe sporophyte/ dispersal ofspores. Thisprocess is facilitatedby elaters, gametophyteinterface, is ubiquitousin embryophytes specializedelongated sterile cells withspiralled wall (Ligrone et al. 1993),this specializedabsorptive region thickenings.With anephemeral,elongated, mature sporo- wasprobably in place in the earlystages ofsporophyte phyteit followsthat there wouldbe no selection for diversi¢cation. The occurrence oftransfer cells ingame- elaborationof conducting tissue, photosyntheticregions tophytetissue surroundingthe zygoteof Coleochaete andstomata in liverworts.Such tissues andcells areadap- supports this supposition.Thus, certain Charophyceae tationsbene¢ cial to sporophytes, such asthose ofmosses werepreadapted for embryo evolution (Graham 1993).As andhornworts where sporophyte extension is gradualand anunelongated mass ofcells, the ancestralsporophyte broughtabout by cell divisionfollowed by gradual elon- wouldnot have acquired structures necessary for gation.Mechanical strength andmovement of materials, sustained growth,e.g. localized meristems, photosynthetic especiallyupwards to the regionwhere spores di¡er- zones,conducting tissue andstomata. In particular , entiate, arecrucial to the success ofsporophytes that stomata,assimilative regions and conducting tissues are persist throughthe growingseason and disperse spores adaptivespecializations that enablepersistence ofan elon- overan extended period. Hence, selective pressures inat gatedsporophyte through the growingseason. Therefore least some ofthese lineageshave favoured the retention of the homologyof such structures amongbasal land plants sporophyticinnovations related to increased strength requires evaluation. (cells withthickened walls) ,photosyntheticfunction Stomataoccur in Sphagnum andthe true moss lineage (stomata andassimilative regions) and enhanced trans- (but notbasal taxa such as Takakia andAndreaeales) and portof nutrients (conductingtissue) .Sporophytesof hepa- intwo of the ¢vewidely recognized genera of hornworts. tics require acomparableperiod of time tomature but Itshouldbe noted that Megaceros,the hornwortgenus this process occurs withinthe con¢nes ofprotective withthe largestsporophytes, does not possess stomata.In gametophytictissues whichprovide a continuoussupply of manymosses, insteadof distinct guardcells, stomate waterand nutrients. ontogenyuniquely produces a binucleatecell witha Notinsigni¢ cant in e¡ ecting spore dispersal is the fact central poreformed by incomplete septum formation that manybryophytes grow on vertical substrates. Insuch duringcytokinesis (Sack & Paollilo1985) .Separationof situations,the entire plantis elevatedabove ground level adjacentcells alongthe middlelamella is commonplacein andis exposedto wind currents that facilitatemaximum hornwortgametophytes. I twouldfollow then that the sporedispersal. Sporophytes of epiphytic mosses, for geneticpotential for producing two adjacent cells that example,are often less elongatedthan those ofground separatefrom each other, i.e. stomata, is inherent inthese dwellingtaxa. Thus, location of habitat must befactored plants.Since this innovationis restricted inoccurrence to intothe equationfor successful spore dissemination. onlyone lineage of anthocerotes ( Phaeoceros^Anthoceros), Di¡erent strategies ofsporophyteelevation are comple- stomataprobably evolved independently in mosses and mented bydivergent morphogenetic designs in embryo hornworts.This interpretation is reasonablegiven the andsporophyte growth and di¡ erentiation. Salient simplicity ofstomatal design and the apparentlack of features ofsporophyte development in bryophyte lineages diurnalcycles inbryophyte stomata (Paton & Pearce areconsidered below .Thehornwort zygote is dissected 1957).Certainly,much more complicatedstructures and ¢rst bya verticalwall, not a transverse partitionas in processes haveevolved multiple times inembryophytes;

Phil.Trans.R. Soc.Lond. B (2000) Vegetative and reproductive innovations ofearly land plants K. S.Renzaglia and others 783 secondarytissues andheterospory are two excellent containedwithin, the sporodermwas a necessary adapta- examples(Bateman et al. 1998;Niklas 1997) .Bateman& tionfor survival and dispersal ofairborne spores. Intine DiMichele (1994)concluded that heterosporyevolved a andexine are derived from the cytoplasmand are minimum of1 1times duringembryophyte history . depositedin a centripetal fashion.T ripartite lamellaeare Amongbryophytes, conducting tissues arerelatively majorcomponents of the exinein most mosses, liverworts widespreadin gametophytes but restricted indistribution andtracheophytes, but apparently are lacking in horn- withinsporophytes to mosses, afeaturethat suggests a¤- wortsand Andreaeales (Brown & Lemmon 1988;R enza- nities withtracheophytes. However, because of develop- glia& Vaughn20 00).Aprimexineis laiddown during mentaland ultrastructural divergences,contemporary meiotic cytokinesisin liverworts and hornworts and this investigationshave called into question the homologyof layerprovides a template withinwhich wall layers are moss andtracheophyte conducting cells andhave resulted successively deposited.Premeiotic patterningof wall sculp- inthe rejection ofthis presumption (see Ligrone et al., turingis restricted tocertain liverworts( Haplomitrium , this issue andreferences therein).Most features ofsieve Pallavicinia and Nowellia) (Brown et al.1986);with a more cells, the food-transportingcells ofpteridophytes, which comprehensivesurvey ,this featuremay be informative in areshared with leptoids, the food-conductingcells of de¢ning clades within hepatics. W all-sculpturingpatterns mosses, arefound exclusively in the Polytrichales,a rela- involvingdeposition of aperinefrom extrasporal tissue are tivelyderived moss taxon.The fundamental cytological sharedby mosses andpteridophytes (Blackmore & Barnes design,involving an endoplasmic microtubule system, is 1987).Atrue perine is lackingin hornworts and liverworts, sharedby mosses andliverworts and is absentin tracheo- whereouter exine layers are responsible for wall ornamen- phytes.Similarly ,the conceptof homology between tation.In hornworts, a dense layeris depositedlate in devel- hydroids,water-conducting cells ofmosses, andtracheids, opment(Renzaglia & Vaughn200 0).This`pseudoperine’is water-conductingcells oftracheophytes, has been severely di¡erentiated fromtrue perine becauseit is derivedfrom challenged.Basal moss clades,except Takakia, are devoid the sporemother cell walland not the tapetum. ofconducting cells andthose of Takakia more closely Elaters consistingof entire cells arerestricted toliver- resemble water-conductingcells ofthe liverwort Haplomi- worts andhornworts. These cellular entities arethe trium thanthey do those ofthe true moss lineage.Clearly , productof controlled mitotic divisionsand thus the ratio conducting-cellhomology among bryophytes and ofspores toelaters oftenis ¢xedwithin a taxon.In tracheophytesrequires further evaluationand can no general,the ratioof spores toelaters is greaterthan four longerbe regardedas aworkingprecept. toone in hepatics; mitotic divisionsoccur in sporogenous Theprimitive methodof sporangial dehiscence is the cells after the elater mother cell is delimited. Incompar- formationof a singlesuture. Amongarchegoniates this ison,elaters ofhornwortsare laid down in tiers that alter- conditionis present inall basal clades. The process may natewith spore-generating layers. T ypicallyfour spores involvea specializedline of cells asin Haplomitrium areproduced per elater,butin some instances mitotic (Bartholomew-Began1991 )orit mayoccur between divisionsin the elater mother cell decrease the spore-to- seeminglyundi¡ erentiated cells asin Takakia and Huperzia elater ratio(Schuster 1984 c).Thelack of specialized wall (Renzaglia et al.1997;A.R.Maden,unpublished data) . thickeningsin most taxaand the tendencyfor adjacent Hornwortsporophytes typically dehisce alongtwo sutures elaters toadhere to one another has led to the concept oneither side ofthe sporangium,while capsules of more that the `pseudoelater’of hornworts is multicellular.The advancedliverworts split intofour valves and those of curiousoccurrence ofelaters withspiralled wall thicken- mosses producean operculumwith an underlyingelaborate ings in Megaceros and Dendroceros suggests homologyof peristome. these cells withmorphologically similar elaters ofhepa- Sporesand sporogenesis are fundamentally conserved tics. However,because of the variabilityin elater struc- inembryophytes and provide a wealthof phylogenetically ture inhornworts and the existence ofspiralled elaters in informativecharacters (Brown& Lemmon 1988,1990 b; presumablyderived taxa ( Megaceros and Dendroceros ), the Garbary& Renzaglia1 998).Forexample, monoplastidic resemblance ofelaters ofcertain hornworts and liverworts meiosis isanancestralcondition that characterizes Coleo- ismost realisticallyinterpreted asa convergence.F urther chaetales,hornworts, mosses andprimarily basal taxa of developmentalstudies arerequired totest this hypothesis. liverworts,lycophytes and ferns (Renzaglia et al. 1994; Brown& Lemmon 1997).Associatedwith monoplastidy 7.PHYLOGENY INTERPRETED FROM MALE inarchegoniates, but not in greenalgae, is auniquequad- GAMETOGENESIS ripolarmicrotubule system (QMS) that is organizedat the plastids andpredicts the polarityof the twomeiotic Acomprehensivedata set onspermatogenesis wasdevel- divisions(Brown & Lemon1997) .Theoccurrence ofa opedto address relationshipsamong pteridophytes and QMS inboth monoplastidic and more-derived polyplas- bryophytes(Garbary et al.1993).Theoriginal data set tidic meiocytes ofliverworts and the putativeloss ofthis consisted of9 0characters. Accumulationof additional microtubuleassemblage in tracheophytesprovides a well- developmentaland experimental data from other organ- de¢ned cytomorphogenetic transformation series inland isms hasresulted ina deeper understandingof character plants(Brown & Lemmon 1997). homology,andthe original90 -character dataset hasnow Thespore wall(sporoderm) ofembryophytes shows beenreduced to 72 more informativecharacters. Thus, continuityin structure, typicallywith a pecto-cellulosic datafrom male gametogenesis may provide the singlebest innerlayer (intine) andan outer,sculptured layer(exine) , set ofmorphological characters interms ofhomology . whichis impregnatedwith sporopollenin (Blackmore & Recent e¡orts toincrease taxonsampling and to complete Barnes1987) .Asaprotectivecoating around the singlecell dataon critical pteridophyteand bryophyte genera have

Phil. Trans.R. Soc.Lond. B (2000) 784K. S.Renzaglia and others Vegetative and reproductive innovations of early land plants

Figure10. One of threetrees of length164 (CI 0.78, RI 0.88)obtained from a maximum ˆ ˆ parsimonyanalysis (implemented with PAUP*4.0) ofcharactersderived from spermatogenes is. Numbersabove each branch represent the number ofcharacterssupporting the branch. Bootstrap percentagevalues (100 replications) followed by decayindices are given below the branch. enabledgreater resolution of relationships, especially those basedon rbcL (WikstrÎm &Kenrick1 997;Korall et withinlycophytes. Of specialimportance were the archi- al. 1999),exceptfor the placementof Phylloglossum in a tectural anddevelopmental data for Selaginella kraussiana clade with Lycopodium, not Huperzia.However,relation- (Renzaglia et al. 1998) and S.australiensis (Renzaglia et al. ships amongthese three homosporoustaxa are poorly 1999);previous data were derived mainly from the mature resolvedbased on spermatogenesis. Becausethere is enor- sperm structure (Robert 1974).The1993 analysis resulted mous variationin male gamete structure withinlyco- inahighlyintuitive cladogram with bryophytes monophy- phytes,it wouldbe useful toexpand the dataset to letic andwith mosses andliverworts forming a single includeall generic segregates oflycophytes (Ò llgaard clade.The only apparent anomaly was the placementof 1987)and to further samplesubgenera within Selaginella . Selaginella onthe bryophyterather thanthe tracheophyte Theplacement of Equisetum inthe fern lineageis contro- clade.Subsequent cladistic analyses of this sperm dataset versial(Kenrick &Crane1 997 a,b); however,the ¢rst resolvethe anomalousposition of Selaginella (Maden et al. unequivocalevidence that supportedthe hypothesisthat 1997;this paper),andplace it withother lycophytes. horsetails arereduced ferns camefrom male gametogen- Thecurrent maximumparsimony (MP) analysisof 2 1 esis characters (Bierhorst 1971).Thishypothesis later found archegoniatesusing 72 characters producesthree equally supportin ananalysiscombining rbcL andmorphological parsimonioustrees that havethe same basictopology characters (Pryer et al. 1995)as well as an analysis of (¢gure 10) as those publishedpreviously (except forthe mtSSU rDNA sequences (Du¡& Nickrent 1999).Asister more appropriateposition here of Selaginella with the relationshipbetween Psilotum andeusporangiate ferns is lycophytes),inwhich there is adichotomybetween bryo- supportedby a number ofmolecular studies (Manhart phytesand tracheophytes. The monophyly of bryophytes 1995; Pryer et al.1995;Pahnke et al.1996;Malek et al. 1996; receives weakbootstrap (BS) support(68% BS, decay Wolf1997 ;Wolf et al.1998;Hedderson et al. 1998). indexof 2) ,whereasthe tracheophyteclade is strongly Manualadjustments ontree topologywere conducted supported(97% BS, decay index of 5) .Within bryophytes, usingMacClade (Maddison & Maddison1 992)to study allthree groupsare monophyletic and the moss andhorn- the e¡ect ontree length(TL) .Onlytwo other hypotheses wortclades are strongly supported. There is onlyweak werereasonably consistent withthese data.T ree lengthwas supportfor the moss plusliverwort clade (BS 57%,decay onlytwo steps longer(TL 166)when ¢ gure10 was ˆ indexof 1 ); however,three fundamentalcharacters constrainedto match the results fromthe overallmorpho- supportthis clade,i.e. dimorphic basal body structure, logicaldata set (¢gure 1 1)andthe moleculardata set the presence ofa splineaperture andthe locationof the (¢gure 1 2),bothof which resolve hornworts as sister toall stellate pattern inthe £agellum. other embryophytes.The embryophyte clade minus horn- Lycophytesform a cladesister toa monophyleticassem- wortsis supportedby a characterassociated with the stag- blagecontaining ferns, Equisetum, Psilotum andseed plants. geringof the basalbodies, a fundamentalfeature of motile Theclade of heterosporous taxa ( Isoe« tes and Selaginella ) is cell organization(R enzaglia& Duckett 1991;see discus- stronglysupported (BS 94%,decay index of 4) ,whereas sionin ½ 5).Anadditionalsingle step (i.e. toTL 167) was ˆ its sister group,containing the remaininghomosporous required tomake mosses sister totracheophytes while lycophytes,receives weakersupport (BS 60%,decay retaininghornworts as the basallineage. The moss/ indexof 1 ).Thisanalysis of lycophytes is congruentwith tracheophyteclade is supportedby two characters: the ¢rst

Phil.Trans.R. Soc.Lond. B (2000) Vegetative and reproductive innovations ofearly land plants K. S.Renzaglia and others 785

Figure11. The singlemost parsimonious tree of length264 (CI 0.70, RI 0.84)obtained from ˆ ˆ amaximumparsimony analysis (implemented with PAUP* 4.0)of 125developmental and morphologicalcharacters.Numbers above each branchrepresent the number of characters supportingthe branch. Bootstrap percentage values(100 replications) followed by decayindices aregiven below the branch.

involvesspline growth (in associationwith nuclear provideabundant data for a robust reanalysisand will shaping)and the secondinvolves the directionof nuclear enablere¢ ned evaluation of preadaptations in these cells shaping.Other tree topologieshad even less support,and that ledto the architecture ofsperm cells ofbasal whenthe liverwortswere placed as the outgroupto the embryophytes. remainingland plants (i.e. the Mishler &Churchill( 1984) Our MP analyseshave dealt only with land plants with hypothesis),the cladogramwas ¢ vesteps longerthan the £agellatedsperm. Thecomparison of £ agellatedand non- most parsimonioustree (TL 169).Forthis arrangement, £agellatedsperm bySouthworth & Cresti (1997)suggests ˆ there wereno characters that supportedthe clade that analogousfeatures maybe present innon-£ agellated consistingof allembryophytes minus liverworts. seed plants,and that there aresome remnants ofstructural Thelack of congruence between the tree topology homologybetween the twoarchitectures. However,there basedon spermatogenesis (¢gure 10) and that basedon maybe too few characters onwhich to attempt aphylo- overallmorphology (¢ gure 1 1)maybe due to inherent geneticanalysis across the transitionbetween these sperm problemsin the spermatogenesis dataset. First, because types. Theabsence of comparable studies ofspermato- ofconvergences in cell architecture betweenspermato- genesis ingymnosperms and basal angiosperms is alsoan zoidsof Charalesand archegoniates, Chara may not be an impediment toa critical analysisof non-£ agellated sperm idealchoice for an outgroup. However, spermatogenesis evolution. in Coleochaete ,anotherpotential charophyte outgroup, is insu¤ciently known, hence many characters cannotbe 8.PHYLOGENY INTERPRETED FROM codedfor this genus.Moreover, based on the limited data MORPHOLOGY AND DEVELOPMENT available,spermatozoids of Coleochaete lackthe presumed ancestralfeatures ofarchegoniate sperm cells. For Acomprehensivedata set incorporatingmorphological, example,no coiling has been described incoleochaeta- developmentaland ultrastructural features wascompiled leanspermatozoids. One couldargue that the selective toapproach questions relatingto the positionof the three pressures formotile cells ofland plants are decidedly bryophytegroups within the globalphylogeny of plants di¡erent fromthose confrontingaquatic algae and thus (Garbary& Renzaglia1 998).Speci¢c characters toresolve evolutionof these cells inarchegoniates was rapid and relationshipsamong groups of tracheophytes generally directional.Moreover ,multicellulargametangia and the werenot included. Of the 125characters, 62were coded problemsrelated to negotiating passage through an forthe gametophytegeneration and 63 werecoded for the archegonialneck provided additional selective forces. As sporophytegeneration. T ocurtailoverweighting of charac- notedabove, similarities inarchitecture ofcharalean and ters relatedto spermatogenesis, only1 1informative bryophytespermatozoids may re£ ect similar structure of features ofmale gametogenesis were included. T ohelp the oogoniumwith its crowncells andthe archegonium ensure characterhomology ,features ofgametophytes were withits neckcells andthe commonconstraints ofmove- consideredindependent of sporophytes. ment througha narrowchannel. Theanalysis presented here of22 ingroup taxa repre- Giventhe results ofour successive MP analysesof the sents aslightlyrevised characterand organism matrix spermatogenesis dataset, weregard the occurrence of relativeto Garbary & Renzaglia( 1998).Asinglemost bryophytemonophyly as an artefact. However ,weare parsimonioustree results fromMP analysisof the convincedthat more detailedanalyses of sperm cell ultra- morphologicaldata (¢ gure 1 1).Thistree is highlyresolved structure ina widerange of charophycean algae will asshownby the factthat 13ofthe 20totalingroup clades

Phil. Trans.R. Soc.Lond. B (2000) 786K. S.Renzaglia and others Vegetative and reproductive innovations of early land plants

Figure12. The singleMP treeof length 2543 (CI 0.67, RI 0.53)resulting from a combined ˆ ˆ analysisof nuclearand mitochondrial SSU rDNA. Numbersabove each branch represent the number ofcharacterssupporting the branch. Bootstrap percentagevalues (100 replications) followed by decayindices are given below the branch. Nodes A andB receivedBS supportof 55%and 61%, respectively,when theoutgroup Prototheca was removed.Taxa with anasterisk(*) lackmitochon- drialSSU rDNAsequences. receive BSsupportof 70% or greater.Strong BS support otherphylogenetic hypotheses. Thus, forcing bryophyte exists forthe basalposition of hornworts, the monophyly monophyly(i.e. the spermatogenesis hypothesis)produces ofhornworts, mosses andliverworts, the moss/liverwort acladogramthree steps longer(TL 267);making ˆ clade,tracheophytes and ferns. Amongthese primary mosses sister totracheophytes adds four steps (TL 268); ˆ clades,only the separationof lycophytes from other reproducingthe Mishler &Churchill( 1984)hypothesis tracheophytesreceived weak support (51% BS, decay (i.e. liverwortssister toremaining embryophytes) was indexof 1 ).Theremaining weakly supported nodes exist sevensteps longer(TL 271).Thusthe totalmorphology ˆ mainlywithin the moss andliverwort clades. Despite tree supports the primaryfeatures ofthe tree from di¡erent taxonsampling, this phylogramis generally spermatogenesis. Basedon the natureof the characters congruentwith the analysisof spermatogenous characters supportingthe primaryclades we consider the cladogram (¢gure 10) with the followingthree exceptions:(i) the in¢ gure1 1tobe an accurate representation ofrelation- positionof hornworts as sister tothe remainingland ships atthe baseof the landplant clade. I tis also plants;(ii) the relativepositions of Psilotum and Equisetum; congruentwith previously published trees basedon mole- and(iii) the resolutionof relationships within the moss culardata (see ½9). clade.The two primary features ofthis cladogramare the positionof hornworts basal to all other land plants and 9.PHYLOGENY INTERPRETED FROM NUCLEAR the positionof mosses andliverworts as monophyletic AND MITOCHONDRIAL SSU rDNA andsister totracheophytes. Thecharacters supportingthe individualclades are Totest hypothesesbased on morphology and to eval- largelydiscussed byGarbary & Renzaglia( 1998)and uatemorphological character evolution, we conducted willnot be repeated here. Theprimary branching within analysesof 22 land plants using a dataset combining landplants (i.e. embryophytesminus hornworts)is sequences fromnuclear and mitochondrial SSU rDNA. supportedby four synapomorphies :(i) the changefrom a ThisMP analysisincludes previously published sequences dorsiventralto an axial gametophyte ;(ii) the presence of inaddition to new sequences including Psilotum, Megaceros £agellarstaggering ;(iii) the presence of£ avonoids;and aenigmaticus , Polytrichum and Cycas formitochondrial SSU (iv)the occurrence ofgrana end membranes. Theposi- rDNA and Megaceros tosanus fornuclear SSU rDNA (see tions of Megaceros and Takakia areof interest asthese taxa Du¡& Nickrent (1999)for details on methods) .Mito- lackstomata and they are the apparentbasal clades of chondrialSSU rDNA sequences werenot available for bothhornworts and mosses, respectively.Thisprovides Spirogyra, Chara, Coleochaete and Selaginella ,sothese taxa strongsupport for our contention that stomatamay have werescored as`missing’ inthe datamatrix. The single evolvedseveral times withinland plants. A similar tree tree resulting froma heuristic search of3647 characters topologyis supportedby our molecular analysis (below) (562informative) shows the same basictopology as the withrespect tothe positionof Takakia butnot the rela- totalmorphology tree (¢gure 1 2).Thetree iswell-resolved tionshipswithin hornworts. with1 4ofthe total2 1ingroupnodes receiving BS support Manipulationsof the cladogramin ¢ gure1 1using of70% or more. StrongBS supportexists forthe mono- MacCladedo not provide even weak support for any phylyof hornworts, mosses, liverwortsand ferns, but

Phil.Trans.R. Soc.Lond. B (2000) Vegetative and reproductive innovations ofearly land plants K. S.Renzaglia and others 787 unlikethe morphologicaltree (¢gure 1 1),onlyweak chondrial cox3 gene.Steinhauser et al. (1999)and Beckert supportis obtainedfor the basalposition of hornworts et al.(2000)arrived at the same conclusionbased on the amongland plants (BS 43%),amoss ^liverwortclade (BS jointpossession ofa groupI intronand a phylogenetic 36%)and tracheophytes (BS 45%).Itis ofinterest that analysisof mitochondrial nad5 sequences. Arecent study BSsupportfor the tracheophyteclade ( Ain¢ gure1 2) employing¢ vegenes from the chloroplastgenome (but increases to55% and the lycophyteclade (B in¢ gure1 2) withlimited taxonsampling) also derived the same basic increases to61% when the distant outgroup Prototheca is tree topology(Nishiyama & Kato1 999).Althougha removedfrom the analysis.Support for these andother number ofmolecularanalyses and biochemical characters relationships,including the basalposition of hornworts, is haveproduced conclusions that arenot consistent with greatlystrengthened bythe additionof mitochondrial ourtree (reviewedby Qiu &Palmer1 999),these were SSUrDNA sequences ofalgal outgroups that aremore oftencompromised by small numbers oftaxa, short genes closelyrelated to embryophytes than Prototheca (e.g. Chara) (orincomplete sequences) ,saturatedthird positionsin (C.L. Parkinsonand J .D.Palmer,unpublished data) . protein-codinggenes, rate heterogeneityand problematic Thistree topologyis alsocongruent with the maximum analyses(e.g. V ande Peer et al.1990;W aters et al. 1992; likelihoodtree derivedfrom the mitochondrialSSU Manhart1 994;Mishler et al.1994;Bopp & Capesius rDNA dataalone (Du¡ & Nickrent 1999).Additional 1996; Lewis et al.1997).Thus,we do not consider them analysesutilizing more genesand greater taxon sampling compellingrefutations of our primary conclusions. The pointto even stronger supportfor both the basalposition distributionof mitochondrial introns discussed byQiu et ofthe hornwortsand the sister relationshipof liverworts al. (1998)supports liverwortsas the basalmostland plant andmosses (D.L.Nickrent, R. J.Du¡, C.L. Parkinson lineage,and is thus inconsistent withour conclusion. I t andJ .D.Palmer,unpublished data) . shouldbe noted that their dataset hasa highlevel of Despite poorbootstrap support for some critical basal homoplasyas several lineages higher in the cladogram cladesin the present analysis,rearrangements ofmajor alsopossess the same characterstate asliverworts. Thus, branchesin ¢ gure1 2tomatch otherproposed topologies the absenceof these three introns canbe explained as result ineven weaker support. F orexample, a cladogram arisingfrom a loss inthe commonancestor to liverworts. congruentwith the Mishler &Churchill( 1984)hypoth- Givenour limited understandingof mitochondrial esis is sixsteps longerthan the MP tree, anda monophy- genomeevolution in plants, we suggest that phylogenetic letic bryophytetopology is eightsteps longer.Ithas relationshipsbased on mitochondrial introns beinferred becomeapparent that the phylogeneticsignal present in withcaution. nuclearSSU rDNA is low,especiallyfor deeper nodeson the landplant tree (Soltis et al. 1999);thus this partition 11.INFERENCES ABOUT LAND PLANT EVOLUTION contributes asigni¢cant amount of homoplasy to a combineddata set. Itis premature toenumerate detailedspeculations on morphologicaltransformation series inbryophytes. A number ofhypotheses have been laid out in the above 10.CONGRUENCE OFMORPHOLOGICAL AND discussion andthese willrequire rigoroustesting asrobust MOLECULAR PHYLOGENIES molecularand total evidence analyses on more taxa Thethree separatedata sets arelargely congruent and becomeavailable. In this section wemake limited infer- pointto a singletopology for the relationshipsof the major ences aboutcharacter evolution based on the consensus groupsof extant embryophytes. The key features ofthis topologyoutlined in }10. phylogenetichypothesis are as follows:(i) thathornworts, Monosporangiatesporophytes and continuity in game- liverworts,mosses, lycophytesand the remainingpterido- tophytefeatures such asapicalorganization and gametan- phytesand seed plantsare each monophyletic; (ii) that gialdevelopment support bryophytes as basal land plant hornwortsare sister tothe remainingland plants; (iii) that lineages.Thalloid gametophytes occur in hornworts and as mosses plusliverworts are monophyletic; and (iv) that this such arebest viewedas plesiomorphicamong land plants. moss/liverwortclade is sister toanother comprising Earlypatterns insporeling development suggest that an tracheophytes.Of these features it is the basalposition of axial,erect habitis ancestralto the prostrate growthform the hornwortsand the sister grouprelationship between ofmanymature liverwortand pteridophyte gametophytes mosses andliverworts that seem tobe most debated. (Whittier &Braggins1992; Bartholomew-Began 1991 ; Becauseof their universaloccurrence, SSU rDNA Bartholomew& Crandall-Stotler1 986).Leafybuds that geneshave been utilized extensivelyin phylogenetic originatefrom moss protonemataare also upright. Thus, studies ofthe livingworld, and speci¢ cally nuclear SSU the moss/liverwortprogenitor was probably a branched, rDNA sequences areinformative in reconstructing plant, upright,lea£ ess axis.Lateral appendages, `leaves’and the algaland protist phylogenies(Soltis &Soltis1 998;Soltis procumbenthabit apparently evolved a number ofinde- et al. 1999).Our primaryconclusions are congruent with pendenttimes ingametophytes of mosses andliverworts. nuSSUrDN Aphylogenies(e.g. Hedderson et al. 1998; Parallelacquisition of `leaves’ in such taxaas Takakia, Soltis et al.1999),althoughit shouldbe noted that some true mosses, Haplomitrium , Phyllothallia , Fossombronia and analysesof this genehave given radically di¡ erent results Jungermannialesis supportedby divergent develop- (e.g.Bopp & Capesius1 996;Capesius & Bopp1997) . mentalstrategies aswellas ourphylogenetic analyses. Previousmolecular analyses have also supported the Sporophytereduction has occurred in all three bryo- basictree topologiesshown in ¢ gures 11and1 2.Malek et phytelineages and in our analyses is best illustrated by al. (1996)derived a sister grouprelationship between the hornwort Notothylas.Boththe molecularand morpho- mosses andliverworts by using sequences ofthe mito- logicaltrees supportthe hypothesesthat the highly

Phil. Trans.R. Soc.Lond. B (2000) 788K. S.Renzaglia and others Vegetative and reproductive innovations of early land plants

Selaginella Blasia

Lycopodium

Haplomitrium

Aulacomnium Psilotum

Phaeoceros Angiopteris

Chara Equisetum

Figure13. Sperm cell architecture mapped onto a cladogrambased on totalmorphology and nuclear plus mitochondrial SSU rDNAsequences. See ½ 11for explanation of the¢ gure.Colour coding: red £agellaand basal bodies; blue nucleus; ˆ ˆ brown mitochondria;yellow splinemicrotubules; orange or purple lamellarstrip; green plastid;grey extraneous ˆ ˆ ˆ ˆ ˆ cytoplasm. simpli¢ed sporophyte of Notothylas is derivedfrom the phenomenaand the seasonalinstability experienced by more complicatedsporophytic structure that typi¢es the these plants. group.This concept was promoted by traditional Homologyof stomata is assumed inmany morpholo- morphologists(Schuster 1984 c,1992;Proskauer 196 0; gicalphylogenies (Kenrick &Crane1 997 a,b; Mishler & Lang1 907;Campbell 1 895;Bartlett 1928)but has been Churchill1984, 1 985).Developmental,physiological and calledinto question by more contemporaryneontologists morphologicaldi¡ erences amongstomata of hornworts, (Mishler &Churchill1 985;Graham 1 993). Notothylas mosses andtracheophytes as well as the occurrence of occurs indisturbed sites that presumablyselect forrapid these structures inspecialized and restricted mosses and generationtime andreduced complexity of both life hornwortssuggest that reappraisalof stomate homologyis cyclephases. In general, the rate ofgenomic evolution, warranted.Interpretation ofindependent acquisition of andconsequently phenotypic change, is greaterin small stomatain three lineagesis equallyparsimonious with the organismsthat growand reproduce rapidly ,aphenom- viewthat stomataevolved once in embryophytes and enonthat inturn is oftencorrelated with low genome werelost inliverworts, three hornwortgenera and basal size (Niklas1 997;John& Miklos1 988;Renzaglia et al. mosses. Thespeculation that bryophytegroups diverged 1995).Thus,the interpretationof small ephemeraltaxa priorto elongation and elaboration of sporophytes such as Notothylas amonganthocerotes and Riccia among supports the contentionthat stomata are homoplastic in hepaticsas derived is consistent withlife history these plants;there wouldbe no selective forces favouring

Phil.Trans.R. Soc.Lond. B (2000) Vegetative and reproductive innovations ofearly land plants K.S.Renzaglia and others 789

Phylloglossum

Lycopodium

Huperzia

.. Isoetes Lycopodiella

Palhinhaea Selaginella

Figure14. Lycophyte sperm cell architecture mapped onto a cladogramderived from spermatogenes isand rbcL sequences (WikstrÎm &Kenrick1997). Multi£ agellated spermatozoids have evolved two independent times in lycophytes,once in the heterosporous Isoe« tes andonce in thehomosporous Phylloglossum .Within Lycopodiaceae,bi£agellated sperm cells of Lycopodium and Huperzia areminute and coiled while thoseof Lycopodiella and Palhinhaea aremore ovoid.

stomate evolutionin the unexpanded,ephemeral ances- Scrutinyof sperm cell structure andevaluation of tral sporophyte.Modern comprehensive comparative evolutionarytrends basedon molecular and morpholo- studies arerequired to further test stomate homology gicaltrees revealsexamples of parallelisms, divergences amongbryophytes and basal pteridophytes. andconvergences among archegoniates (¢ gures 13and Ultrastructural dataon food-conductingcells supporta 14).Theplesiomorphic architecture ofplant spermato- moss^liverwortclade and provide refutation of the precept zoidsis aslightlycoiled bi£ agellated cell. Presumably, that leptoidsin mosses arehomologous to sieve cells of increasedcompaction and coiling occurred independently tracheophytes.Because water-transporting cells probably inseveral lineages, including the Charales,mosses, liver- evolvedat least three times inliverworts and twice in worts,heterosporous lycophytes and ferns (¢gure 1 3). mosses (Ligrone et al.,this issue),caremust beexercised in Hornwortsperm cells remainedsmall anddiverged from interpreting homologyof these cells amongtaxa. otherbryophytes before £ agellarstaggering evolved. An

Phil. Trans.R. Soc.Lond. B (2000) 790K. S.Renzagliaand others Vegetative and reproductive innovations ofearly land plants autapomorphyof hornworts is the reverse indirection- From these data,and from combined analyses with alityof coiling, which, as noted above, was probably morphologicaldata, a more resolvedphylogeny should inconsequentialto the hydrodynamicsof this minute cell. emerge that willprobably mirror ourgeneral conclusions. With resolutionof a moss ^liverwortclade, the detailed Suchcongruence will allow the phylogeneticdebate to similarities inlocomotory apparatus microstructure proceedbeyond that ofbranching patterns sothat plant betweenthese twogroups are interpreted ashomologous. biologistscan investigate equally interesting questions in Flagellarstaggering involving dimorphic basal bodies evolutionarybiology ,includingrates andpatterns of andan aperture inthe splinewere features ofthe proto- evolutionarychange. With robust phylogeniesand the typic moss^liverwortsperm cell that further specialized accumulationof new morphogenetic and ultrastructural after historicalseparation of the twolineages (R enzaglia dataon key taxa, we will be in a positionto make more et al. 1995). accurateand precise inferences aboutstructural homolo- Multi£agellated sperm cells evolvedwithin three sepa- giesin basal land plants. rate lineagesof pteridophytes: in Phylloglossum , a homo- sporouslycophyte (Renzaglia & Maden200 0),in Isoe« tes, This workwould not have been possible without technical and aheterosporouslycophyte and in the Equisetum^Psilotum^ conceptualcontributions of presentand previous students, colla- fern^seed plantlineage (¢ gures 13and1 4).Additional boratorsand colleagues, who have enabled the development of specializationswithin the lycophytesinclude decreased ourideas. In particular we notethe involvement of J.G. Duck- coilingand increased £ agellarstagger in taxa with sub- ett,D .P.Whittier,K. C. Vaughn,A. R.Maden,R. Ligrone, terraneangametophytes, and extreme streamliningin S. J.Schmitt, H. D.Gates,K. D.McF arland,D .K.Smith, heterosporoustaxa. Commonalities in sperm cell devel- H.A.Crumand L. E.Graham.This workwas supported by grantsto K.S.R., D.L.N. and D.J.G. from the National Science opmentand organization provide strong support for a Foundation,USA (DEB-9207626,MCB-9808752 and DEB- fern^Equisetum^Psilotum clade,an assemblage that has 9527735),andthe Natural Sciences and Engineering Research beenproposed by other contemporaryanalyses (Kenrick Council,Canada. W ethankJ. B. Bozzolaat the Micro-Imaging & Crane 1997a,b;Du¡& Nickrent 1999). andAnalysis Center, SIUC, and Thomas Johnson for technical assistancein preparingthe manuscript.

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