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Theoriginof alternation of generations inlandplants: afocuson andhexose transport

Linda K.E.Graham and LeeW .Wilcox Department of ,University of Wisconsin, 430Lincoln Drive, Madison,WI 53706, USA (lkgraham@facsta¡.wisc .edu )

Alifehistory involving alternation of two developmentally associated, multicellular generations (sporophyteand ) is anautapomorphy of ( + vascularplants) . dataindicate that Mid ^Late Ordovicianland possessed such alifecycle, and that the originof alternationof generationspreceded this date.Molecular phylogenetic data unambiguously relate charophyceangreen to the ancestryof monophyletic embryophytes, and identify bryophytes as early-divergentland plants. Comparison of in charophyceans and bryophytes suggests that the followingstages occurredduring evolutionary origin of embryophytic alternation of generations: (i) originof ;(ii) retention ofeggsand on the parentalthallus; (iii) originof matrotrophy (regulatedtransfer ofnutritional and morphogenetic solutes fromparental cells tothe nextgeneration); (iv)origin of a multicellularsporophyte generation ;and(v) origin of non-£ agellate, walled . Oogamy,/zygoteretention andmatrotrophy characterize at least some moderncharophyceans, and arepostulated to represent pre-adaptativefeatures inherited byembryophytes from ancestral charophyceans.Matrotrophy is hypothesizedto have preceded originof the multicellularsporophytes of plants,and to represent acritical innovation.Molecular approaches to the studyof the originsof matrotrophyinclude assessment ofhexose transporter genesand members andtheir expressionpatterns. Theoccurrence inmodern charophyceans and bryophytes of chemically resistant tissues that exhibitdistinctive morphologycorrelated with matrotrophy suggests that Early ^Mid Ordovicianor older relevantto the originof land alternation of generations may be found. Keywords: alternationof generations;embryophytes; bryophytes; charophycean algae ; hexosetransporter genesand

phyceans() independently acquired alterna- 1.INTRODUCTION tionof two multicellular generations; so far as is known, Alternationof generations in is generally their closest extantrelatives (tribophyceans and other de¢ned as the occurrence ofa lifehistory in which there ochrophyte/chromophyte/heterokontalgae) lack such a areat least twomulticellular generations, the gameto- lifehistory .Amongmodern , alternation of phyteand the ,linked by unicellular repro- twomulticellular generations occurs onlyin certain ductivestages, namelygametes andspores (¢gure 1 ). orders ofthe ,and is lackingin the Sporesare generated by sporic , whichis the type three othergreen algal classes that includemulticellular ofmeiosis associatedwith alternation of generations forms (namelyT rebouxiophyceae, and (Raven et al.1999).Thisarticle doesnot address `alterna- Charophyceaesensu Graham& Wilcox(20 00)).Evidence tionof generations’that mayoccur in various autotrophic forindependent of alternation of generations in that occurprimarily as unicells (e.g. certain , brownand green algae suggests that it is highly haptophytealgae) ,orin (such asforamini- adaptive.Hypothetical adaptive aspects oflife history feraand fungi) . variationin autotrophs are discussed byBell & Koufo- panou( 1991),Otto &Goldstein (1992),Baillard( 1997) (a) The occurrence ofalternation ofgenerations andBell ( 1997).Areviewof alternation of generations in inautotrophs landplants, with an emphasis onfossil branchedgameto- histories involvingtwo or more alternatingmulti- phytesthat arethought to be linkedto the lifehistories of cellulargenerations have evolved several times among protracheophytesand early vascular plants, is provided photosyntheticprotists (algae)(Graham &Wilcox20 00). byKenrick ( 1994). Forexample, various bangiophycean red algaehave a life historywith two multicellular stages, anda three-stage (b) change andalternation ofgenerations lifehistory appears to be a basic(plesiomorphic) feature Itshouldbe noted that whiletextbook depictions of for£ orideophyceanred algae.Ancestors ofphaeo- alternationof generations in algae and land plants

Phil. Trans.R. Soc.Lond. B (2000) 355, 757^767 757 © 2000The RoyalSociety 758L. K. E. Grahamand L. W.Wilcox Alternation ofgenerations

s e t a l R! l s e n g a a e l l meiospores f a - r s n a n o h a s s n C e n n c t s a n + s a y e t e h n e n a c a e 2n c p g t a e y r e l n y o m e s s h i c h a t t p g v r p x y p i i h r r t o o u h o d c o s a p n s - e l r o i o w w o o o y e r s u l b s s l l n s c e v a e r e r o h r l r o v s a i o a C T U P M e C l h m v sporophyte gametophyte * * *

Figure1. Diagram of alternationof multicellulargenerations. R!indicatesthe occurrence of meiosis.A lifehistory involving spatiallyand temporally separate generations is characteristi c ofseveralgroups of algae. typicallydescribe the gametophyticgeneration as being Figure2. Diagrammatic representation of phylogenetic haploid,and the sporophyticgeneration as diploid, there relationshipsamong various groups of thegreen algae and aremany examples among the algaeof life history phase embryophytes.Asterisks indicate cases of presumed changethat arenot correlated with change in chromo- independentorigin of alifehistory involving alternation of some number (Graham &Wilcox20 00).Forexample, twomulticellular generations. The barindicates the only the nucleiof and of the brown knowncase among green autotrophs of theoccurrence of a Haplospora globosa ()possess the dependent,multicellular sporophyte (alternation of same number ofchromosomes. However, the DNA level generationsthat are not separated temporally or spatially). ofsporophytic nuclei is twice that ofgametophytic nuclei (Kuhlenkamp et al.1993).Inother algae,environmental Table 1. Matrotrophy and associated life history change have factorsare thought to be as, or perhaps more, important led tothe origin ofthree high-diversity, long-lived thanchromosomal level in determining the directionof lifehistory phase change. Environmental e¡ ects are reproductive regardedas possible explanations for cases ofapogamy innovation mechanism (transition tothe sporophytephase in the absenceof gameteproduction and syngamy) and apospory (transi- £orideophycean carposporophyte n/2n cellfusions tionto the gametophyticphase in the absenceof meiosis andspore production) .Inseedless plants,apogamy and embryophytes dependentembryo placental transfer aposporyare also observed, but dosage e¡ ects are cells important.Maintenance of sporophytic growth depends eutherianmammals viviparity complexplacenta onthe presence ofat least twosets ofchromosomes, whereasgametophytic growth in culture doesnot continuewhen four or more sets ofchromosomes are andlater-divergent tracheophytes (Kenrick &Crane present (Bell1991 ). 1997).Itis importantto recognize that alternationof Inhigher plants, there aremany examples of produc- generationsin the KingdomPlantae is distinctive inthat tionof young sporophytes () fromcells other embryonicsporophytes occur in close spatial and thanzygotes (e.g. embryogenesis,somatic temporalassociation with female (or bisexual) gameto- embryogenesisand ) (Harada et al. 1998), and phytes.Plant embryos, including those ofthe simplest the geneticbasis for such variantsfrom the expectedlife liverwortsas wellas derived angiosperms, seem generally historycycling is becomingclearer. Forexample, the tobe nutritionally and developmentally dependent on gene LEAFYCOTYLEDON1 (LEC1), which parentalgametophyte tissues forat least some periodof encodesa transcriptionfactor ,is su¤cient toinduce time inearly development. The presence ofa dependent -likedevelopment from vegetative cells (Lotan embryonicstage is the basis forthe term embryophytes, et al. 1998). commonlyused asasynonymfor Plantae. The occurrence ofalternation of multicellular generations (c) Importance ofsporophyte/ gametophyte coupledwith dependent embryos in all groups of land interactions plantssuggests that these features areautapomorphic Theabove variations having been noted, a lifehistory (uniqueand de¢ ning) features ofembryophytes(¢ gure 2) . involvingalternation of multicellular gametophyte and Multicellularsporophytes do not occur in the charophy- sporophytegenerations characterizes allgroups of extant ceans,the greenalgal lineage most closelyrelated to landplants, which comprise the KingdomPlantae, as embryophytes(Graham 1993),anddependence of the de¢ned by Raven et al.(1999).Members ofthe extant embryonicsporophyte is lackingin most otheralgae that plantkingdom constitute amonophyleticgroup that havealternation of generations. An exception to this includesmultiple lineagesof early-divergent bryophytes generalityis the £oridophyceanred algae,whose

Phil.Trans.R. Soc.Lond. B (2000) Alternation ofgenerations L. K. E.Grahamand L. W.Wilcox759

benoted,however, that fewactual measurements offertil- izationrates havebeen made for red algae.)Asimilar adaptivebene¢ t hasbeen hypothesized to accrue to - less landplants, whose fertilization rates maybe limited byavailability of liquid for transport of£ agellate (Searles 1980).Therest ofthis paperfocuses on palaeontological,neontological and combined approaches tounderstanding the roleof matrotrophy in the originof alternationof generations and the dependentsporophytic embryoof land plants.

2.PALAEONTOLOGICAL EVIDENCE THAT EARLIEST- KNOWN () LAND PLANTS POSSESSED ALTERNATION OFGENERATIONS 1 m m Microfossils ofthe Mid ^Late Ordovicianage, described fromLibyan deposits byGray et al. (1982), Taylor( 1995)and Strother et al.(1996),provideevidence Figure3. A transmissionelectron micrograph of aportionof that the earliest-knownland plants possessed alternation aplacentaltransfer of theliverwort Conocephalumconicum , ofgenerations. These remains includespores arrayedin showingextensive development of wallingrowths. The persistent tetrads. Persistence ofthe tetrads suggests preparationwas high-pressure frozen, a methodthat facilitates preservationof cellmembranes. the presence ofspore walls that werechemically resistant tothe e¡ects ofmicrobial decomposition and diagenesis, asare the -impregnatedspore walls of carposporophytegeneration is nutritionallyand modernland plants, including those ofbryophytes. developmentallydependent on the femalegametophyte. Similarspore tetrads, knownto have resulted from In£ orideophyceansand land plants, the embryonic meiosis, areproduced by the early-divergent(see Lewis et sporophyte’sdependencyhas been described asmatro- al.1997)complex thalloid liverwort Sphaerocarpos . Such trophy(literally ` feeding’),byanalogy to tetrads remainedintact after high-temperature embryonicnutritional and developmental dependence in (Graham &Gray20 01),aprocedurethat to eutheriansand other viviparous metazoans (Graham, some extent mimics the degradativechemical and L. E. 1996).Matrotrophywas a keyinnovation in the physicale¡ ects experiencedby plant tissues duringfossil- diversi¢cation of these three majorclades (£ orideophy- ization.Occurrence ofthe Ordovicianspores intetrads is ceans,embryophytes and eutherians) ,its adaptivenessin strongevidence that theyarose by sporic meiosis, whichas eachcase hypotheticallyrelated to increases infecundity notedearlier ,is ahallmarkof alternation of generations. (table1 ).Ineach case, there is amorphologicalcorrelate Sofar as is known,meiosis iszygoticin all charophyceans ofmatrotrophy öthe ,a regionof specialized exhibitingsexual reproduction (evidence reviewed by cells (tissues inthe cases ofembryophytes and euther- Graham1993) .TheOrdovician evidence suggests ians)that facilitatesmaternal to embryonic nutrient that sporicmeiosis wasan innovationthat occurredat the transfer. dawnof embryophytes. Inembryophytes (as ineutherians) ,there areno inter- Microfossil evidencethat sporicmeiosis coincidedwith cellularconnections linking parental and embryonic originof amulticellularsporophyte generation in earliest- tissues, hencetransport ofsolutes occurs bymeans ofcell knownland plants was provided by Graham & Gray membrane transporters (i.e. is apoplastic).Embryophytic (2001),whodemonstrated that the sporangialepidermis placentalcells typicallypossess elaboratesystems ofwall of Sphaerocarpos ,onhigh-temperature acidtreatment, falls ingrowths,which greatly increase the surface areaof the apartinto monostromatic cellular fragments resembling across whichfacilitated di¡ usion or active the most ancientmulticellular attributed toland transport must occur(¢ gure 3) .Suchcells areknown as plants.The latter ,derivedfrom Libyan deposits of placentaltransfer cells; these mayoccur on one or both Ordovicianage, were associated with spores (Gray et al. sides ofthe generationalgap. Placental transfer cells are 1982).On the basisof morphometric comparisonwith wellstudied atthe ultrastructural levelin bryophytes(see high-temperature acetolysedsporangial ofthe reviewsby Ligrone & Gambardella1 988;Ligrone et al. early-divergentmoss , Kroken et al. (1996) 1993)and in higher plants such as Arabidopsis (Murgia et suggestedthat the Ordoviciancellular scraps represent al.1993).Physiologicalstudies providesubstantial sporangialepidermal remains, andthat theyare the evidencethat the placentaeof bryophytes (Browning & earliest-knownfossils ofthe sporophytegeneration of Gunning1 979 a,b; Renault et al.1992)and £ owering landplants. plants(V anCaeseele et al.1996) in apoplastic Hydrolysis-resistance ofextant sporangial transport. epidermis wasattributed tothe presence ofhighly Provisionof the £orideophyceanembryo (carposporo- insoluble,wall-bound phenolic , on the basis of phyte)with nutrients fromthe maternalgeneration is speci¢c auto£uorescence properties (Kroken et al. 1996). suggestedto be a strategyfor amplifying the productsof Similar,resistant, auto£uorescent materials occurin sexualrecombination where fertilization rates arelimited vegetativecell wallsof various charophycean algae, bythe absenceof £ agellafrom male gametes. (Itshould suggestingthat landplants inherited the capacityto

Phil. Trans.R. Soc.Lond. B (2000) 760L. K.E.Grahamand L. W.Wilcox Alternation ofgenerations

(a) Stage A Anearlystep, occurringin charophyceans, is hypothes- izedto have been transition from to oogamy (productionof £ agellatesperm andlarger ,non-£agellate ),whichalso occurs inland plants. Determination of the directionof character transition is dependenton the existence ofa robust phylogenyfor charophyceans but, to date,phylogenies of charophyceansthat are inferred from variousnucleic acid sequence datahave been highly incongruent.This state ofa¡ airs hasmost likelyresulted fromextinction, leaving us witha sparse extantrepresent- ationof the group.Consequently ,Graham,L. E. (1996) andGraham & Gray(20 01)recommended relianceon 100 m m moleculararchitectural data, such asintron insertion events, generearrangements ormovement of betweencellular , as is alsoadvocated by Qiu & Figure4. micrograph of aportionof afertile Palmer( 1999).Suchdata indicate that isogamousZ ygne- orbicularis thallusthat was subjected to high-temperatureacid matales areearlier-divergent than oogamous Coleochaete hydrolysis.Cell contentshave been hydrolysed, but walls of thetwo large spherical zygotes and their enclosing cortical andCharales (Graham &Gray20 01;Graham& Wilcox cellsare resistant to hydrolysis.Extensive development of wall 2000). ingrowthsin corticalcells (see arrow, for example) is readily However,Sluiman& Guihal( 1999)reported that 18S discernible. rDNA sequence analysissuggests that the oogamous Chaetosphaeridium ,whichis frequentlylinked with Coleochaete onthe basisof morphological similarities (see producesuch compoundsfrom charophycean ancestors. Graham& Wilcox20 00)and rbcL sequence data Resistant, auto£uorescent wallsof the charophycean (Chapman et al.1998),maynot be closely related to green alga Coleochaete that areassociated with zygotes Coleochaete but,rather ,relativelyearly-divergent within haveF ouriertransform infrared(FTIR) spectra that are charophyceans.Other small subunitrDNA sequence similar tothose ofthe decay-resistant wallsof another analysesindicate surprisingly early divergence of - charophycean,the desmid Staurastrum (Gunnison & leans(Huss &Kranz1 997),this di¡ering dramatically Alexander1 975 a,b),butdi¡ erent fromtypical plant from rbcL dataindicating that acladeincluding both sporopollenin(Delwiche et al. 1989). Charalesand Coleochaete is sister toembryophytes Occurrence ofsimilar ,resistant compoundsin wallsof (McCourt et al.1996).Further analysisof the evolution- bothcharophyceans and bryophyte sporangial epidermis aryorigin of oogamy in the charophycean ^ suggests the possibilitythat the Ordoviciancellular lineagemay depend on our ability to map isogamy to remains cited earlier couldbe of charophycean origin. oogamytransition(s) ontoa robust phylogeny. However,the onlyknown example of resistant multi- Aspects ofthe transitionto oogamy that require further cellulartissue incharophyceansis that ofthe thalluscells, studyinclude: (i) originof the complex,multicellular whichform a corticallayer that nearlycompletely encloses gametangiaof and transition from single-celled mature zygotesof Coleochaete orbicularis (Delwiche et al. antheridiain some of Coleochaete tomulticellular 1989).Insuch aninstance, the zygotesare quite conspic- aggregatesof spermatangial and sterile cells that occurin uousand much largerin diameter thanthe surrounding other Coleochaete species (Graham 1993);(ii) changesin cells (¢gure 4); no such enlargedcells occurwith the Ordo- £agellategamete that maybe linkedto transition viciancell scraps. Resistant materials inbryophyte sporan- tooogamy (Duncan et al.1997);(iii) loss of£agellardevel- gialepidermal cell wallsmay be adaptive in protecting opmentat the originof eggcells; (iv)origin of the enlarged developingspores frommicrobial attack; earliest land eggs,¢ lledwith reserves, ofcharaleans; (v) origin of plantsmay also have derived such abene¢t fromtheir the trichogyne,a tubularprotuberance of eggs, whose presence (Graham &Gray20 00).If Ordovicianmicro- distalwall undergoes controlled hydrolysis, allowing fossil scraps dorepresent sporangialepidermis, the plants release ofsperm attractants andsperm entryin Coleochaete fromwhich they were derived possessed amulticellular (Graham 1993);and (vi) the originand chemical character sporophyteand, hence, alternation of generations. ofsperm attractants inoogamous charophyceans and However,fossil remains ofthe placentalregion, repre- bryophytes.A possibleapproach to the ¢nalissue mightbe sentingevidence that plantshad acquired matrotrophy by to examine Coleochaete andcharaleans for compounds that the Ordovician,are so far lacking. areknown to coordinate mating in zygnemataleans (Sekimoto et al. 1993). 3.HYPOTHETICAL STAGES INTHE ORIGIN OF (b) Stage B ALTERNATION OFGENERATIONS IN Retentionof eggs(and zygotes that developfrom them) EMBRYOPHYTES onthe maternal(or bisexual parental) is the next Severalstages (A ^E)inthe evolutionaryorigin of land hypothesizedstep (¢gure 5) ,asthis is anessential plantalternation of generations(¢ gure 5) can be deduced precedent forthe developmentof nutritionaland develop- bycomparison of the reproductivefeatures ofcharophy- mental interactionsbetween generations. Among ceansand bryophytes. charophyceans,examples of egg/ zygoteretention occurin

Phil.Trans.R. Soc.Lond. B (2000) Alternation ofgenerations L.K. E. Grahamand L.W.Wilcox761

(c) Stage C 1. isogamy oogamy Athird postulatedstep inorigin of the lifehistory of embryophytes(¢ gure 5) is the originof matrotrophy , 2. egg- dispersed egg- zygote retained transfer ofnutrients and/ormorphogenetic factors from on gametophyte parentaltissues tozygotes. Such a step is dependenton (n- 2n signalling and morphogenetic responses) the earlierevolution of retained eggs/ zygotes(see above), becausedi¡ usive or degradative loss ofparentally 3. regulated n- 2n transfer of nutrients via secreted nutrients orother compounds would greatly placental transfer tissues—matrotrophy reduce their supplyto progeny (zygotes) that arespatially separate,particularly in an aquatic environment. Conti- 4. unicellular 2 n (zygote) multicellular 2 n guity of Coleochaete parentalcells andzygotes, localized (delay in meiosis) (sporophyte) developmentof elaborate ingrowths on the wallsof corticalcells that arein direct contactwith zygotes, and 5. sporopollenin-walled sporopollenin-walled conspicuouspost-fertilization nutrient storage(in the zygote- naked spores spores formof andlipid) provide circumstantial evidence foroccurrence ofmatrotrophy .Closespatial association Figure5. Hypothesized stages in theevolutionary origin of ofparental tissues whosecells possess typicalplacental matrotrophicalternation of generationsas itoccursin modern transfer (wall ingrowths) and physiological embryophytes.Some proposed mechanisms are shown in evidencefor solute transport across the apoplastic,inter- parentheses.Shading of no.3indicatesthe area of research generationjunction (Browning & Gunning1 979 a,b; thatis described most fully in thispaper. Renault et al.1992)provide evidence that matrotrophy occurs inbryophytes. These data suggest that embryo- phytescould have acquired matrotrophy from charophy- Coleochaete andCharales, facilitated by mitotic production ceanancestors, andthat matrotrophypreceded the origin ofa layerof corticating cells orelongation of spirally ofmulticellular sporophytes. Molecular strategies for twisted tube cells, respectively.Inboth cases, the testing these possibilities arediscussed ina later section of enclosingcells belongto the parentalgeneration this paper. (Graham 1993).InCharales, tube cells undergoextension atthe same time asthe eggcell enlarges,such that (d) Stage D mature eggsare thought to be near the maximalsize Transitionfrom a unicellularzygote (as producedby reachedby zygotes, and fully enclosed before fertilization charophyceans)to production of a multicellulardiploid occurs (Pickett-Heaps 1975).Incontrast, in Coleochaete , sporophytegeneration (as inembryophytes) by repeated the enclosinglayer of parentalcells doesnot develop until mitotic divisionof the zygoteis the nextpostulated step in after fertilization,and zygotes (rather thaneggs) undergo the originof land plant alternation of generations enlargementand storage accumulation. In both cases, as (¢gure 5) .Themechanism most oftenhypothesized for this yetunde¢ ned cell ^cell signallingprocesses probablyco- transformationis `delayin meiosis’ .Thismeans that a ordinatedevelopment. phaseof mitotic proliferativegrowth would occur between Only in Coleochaete couldinitial development of corti- zygoteformation and spore production by meiosis. An catingcells besaid to result fromintergenerational alternativeidea, that the landplant sporophyte originated (zygote/parentalthallus) communication, but zygote/ ingreen algal ancestors that had¢ rst acquiredalternation parentalcell signallingthat in£uences maturationof the ofmulticellulargenerations, is currently less favoured,as it 2n/n cell complexmay occur in both Coleochaete and Char- isnotsupported by modern phylogenetic analysis. Extant ales.Hence these arethe taxaof choice for analysis of greenalgae that exhibitalternation of generationsare not hypothesizedcommunication systems, di¡usible signal- closelyrelated to the ancestryof landplants, and charophy- lingmolecules andtheir receptors. Some Coleochaete ceangreen algae, which are closely related to embryo- species producezygotes that areless completelycorticated phytes,lack alternation of generations,so far as is known. thanothers (Szyma¨nska1989); comparison of expression Hypotheticalorigin of embryophytes from green algae patterns atcritical developmentalstages mightreveal havingtemporally and spatially separate generations also geneticdi¡ erences relevantto the zygoteretention issue. doesnot provide a satisfactoryexplanation for the originof Eggsare reportedly not retained in Chaetosphaeridium matrotrophy. (Thompson1 969);if Chaetosphaeridium is sister to The¢ ndingof an extant or fossil `charophycean’that Coleochaete ,di¡erential expression studies ofthese taxa hadintercalated even a singlemitotic divisionbetween couldbe veryinformative. syngamyand meiosis wouldmean discovery of a possible Molecules knownto have signalling functions in transitionalform, a descendantof such atransitional higherplants that couldalso operate in charophyceans formor a closeparallel to the simplest possible(two- andearly-divergent bryophytes include celled) landplant sporophyte. The adaptive advantage of peroxide,a di¡usible and relatively long-lived molecule havinga multicellularsporophyte is that greaternumbers whoseproduction is inpart regulated by peroxisomal ofgenetically diverse meiospores couldresult, facilitating catalase(Karpinski et al. 1999);secreted or bothcolonization e¡ ectiveness andincrease inpopulation small proteins that actas (Gehring 1999; geneticvariability ,andhence greater evolutionary £ ex- Fletcher et al.1999);and (Koch 1 996;Graham, ibility.Evidencethat such adaptiveadvantage exists is I. A.1996).Therole of sugars is discussed belowin providedby progressive increase insporophyte size more detail. duringthe course ofembryophyteevolution.

Phil. Trans.R. Soc.Lond. B (2000) 762L. K.E. Grahamand L. W.Wilcox Alternation ofgenerations

Reconstructionof the critical molecularevents involved zygotes sporocytes indelay of meiosis maybe possible by probing the genomesof extant charophyceans and early-divergent CA embryophytesfor genes that areinvolved in the regula- CE tionof meiotic initiation.Such genes have been identi¢ ed 2n 2n 2n inmetazoans (e.g. Barton & Kimble 1990;de V ries et al. SP 1999), (e.g.Englebrecht & Roeder 1990;Matsura et al.1990;Printz et al. 1995; Iino et al.1995;Colomina et 1. gain SP 2. lose SP al.1999;de los Santos & Hollingsworth199 9;Edelmann et al.1999;Ohno & Mattaj 1999;Sagee et al. 1999), and n £oweringplants (e.g. Bouchard 1 990;Bai et al. 1999); a n recent reviewof meiotic chromosomeorganization and n segregationin plants is providedby Dawe ( 1998).Given such information,it mayultimately be possible to (a) (b) (c) transform charophyceansso that their zygotesrecapitu- Figure6. Hypothetical transformatio nsinvolvedin theorigin latethe meiotic delaypostulated to have occurred during ofwalled,non-£ agellate spores, as theyoccur in modern the originof embryophytes. embryophytesby gain of sporopollenin(SP) in sporewalls Theorigin of the quadripolarmicrotubular system (1),then loss of sporopolleninfrom sporocyte walls (2). (QMS)characteristic ofembryophytic sporocytes, which (a)The extantcharophycean Coleochaete produceszygotes providesan essential sca¡olding for proper spatial segre- havingcallosic (CA), cellulosic(CE) and SP layers,within gationof andnuclei into the fourdaughter which develop£ agellate,non-walled meiospores. ( b) A products(Brown & Lemmon 1997),isunknown.Whether hypotheticalintermediate stage in which diploidsporocytes this structural componentof meiotic cells is present inan (thatare homologous to charophyceanzygotes) have resistant walls,and produce walled, non-£ agellate meiospores. earlier formin charophyceans is notknown, and would Persistenceof the resistant sporocyte wall forms an envelope require ultrastructural and£ uorescence immunolocaliza- aroundmeiospore tetrads, preventing their dissociation. ( c) tionstudies ofgerminating zygotes. Correlative analysis Sporopollenin-walled,dissociated meiospores as theyoccur in ofnuclear DNA levelswould also be valuable but has mostembryophytes. beendi¤ cult becauselarge amounts of storage materials, auto£uorescent sporopollenin-likezygote wall layers and sporopollenin-coatedwall of embryophytic spores has auto£uorescent corticalcell walls(in the case of beenregarded as having an adaptive function in the Coleochaete )caninterfere. terrestrial environment,by providing structural stability Microarraystudies inyeast systems haverevealed that andretarding microbial degradation during dispersal ploidychange regulates the expressionof genes involved in (Graham &Gray20 01). the (such asG 1cyclins)and control of Itis asyet unclear whether multicellular sporophytes polarizedgrowth. However, the mechanism bywhich appearedprior to the originof walled meiospores, orvice ploidychange is sensed isnotas yetunderstood (Galitski et versa.I tispossiblethat earliest embryophytes(de¢ ned by al.1999).Sincehomologous cell cycleand actin-regulation presence ofa multicellularsporophyte, however small) genesare likely to occur in plants, it ispossiblethat ploidy mighthave lived in water or very moist environmentsin changesoccurring at the transitions betweengametophytic whichselection didnot operate heavily against unwalled, andsporophytic phases of the lifehistory are similarly £agellatemeiospores. Productionof greaternumbers ofor importantin determiningdi¡ erences inthe morphologyof more geneticallydiverse meiospores couldhave provided the twostages. su¤cient adaptiveadvantage for delay in meiosis tohave occurredprior to origin of walled spores. Ampli¢cation (e) Stage E (apparentlyby reduplication) of zygotic DNA levelsand A¢nalstep, fromproduction of non-walled, £ agellate subsequent productionof 8^32 meiospores in Coleochaete meiospores, asoccurs inmodern Coleochaete ,toproduction (Hopkins& McBride 1976)provides evidence of the ofnon£ agellate, sporopollenin-walled meiospores, as adaptivevalue that amulticellularsporophyte could have occurs inall embryophyte groups, is alsopostulated to innearshore . havebeen involved in the originof plant alternation of Aninner wall layer of sporopollenin-like material is generations(¢ gure 5) .Thisis becausethe relative typicallyproduced during zygote maturation in e¤ciency of walled-spore dispersal andgermination charophyceans.The transition to walled meiospores is success onland may have driven evolutionary transition postulatedto have involved a changein the timingof toincreasingly larger multicellular sporophytes capable of sporopolleninproduction, such that it occurs ata later producingmany meiospores froma singlefertilization developmentalstage, during spore maturation (¢ gure 6) . event.The alternative, illustrated bycharaleans, is Transitionalforms mayhave produced sporopollenin productionof many large, resistant zygotes(representing bothat the sporemother cell stage(hypothesized to be multiple fertilizationevents) ,eachof which produces homologousto charophycean zygotes by Graham 1 990) (probablyonly) one, non-resistant, meiotic product.Such andduring spore maturation. Information regarding areproductivestrategy would not be adaptive if fertiliza- regulationof sporopollenin synthesis anddeposition tionfrequency is limited bywater availability .If spores derivedfrom analysis of higher plant mutants maybe lackingprotective walls were unable to survive to usefulin deducing the genetictransformations that were germinationfollowing terrestrial dispersal, the multi- involvedin the productionof sporopollenin-walled cellularsporophyte likewise would not be adaptive. The spores.

Phil.Trans.R. Soc.Lond. B (2000) Alternation ofgenerations L. K.E.Grahamand L. W.Wilcox763

desirable.I tmaybe possible to determine ifparental 4.APPROACHES TOANUNDERSTANDING OFTHE imprintinga¡ ects allocationof resources atthe plant EVOLUTIONARY ORIGIN OFMATROTROPHY AND placenta(re£ ecting intragenomic, i.e. paternal maternal, PLACENTAL FUNCTION INEMBRYOPHYTES ^ con£ict) ,asis believedto occur in eutherians (Haig Combinedneontological and palaeontological approaches, 1996a,b,1997)and, if so, when this mighthave evolved. andcomparative studies ofextant charophyceans and bryophytesare recommended forelucidation of the evolu- tionaryprocess surroundingthe originof embryophytic 5.COMPARATIVE STUDY OFCHAROPHYCEAN/ matrotrophyand placental function. An example of a EMBRYOPHYTE HEXOSE TRANSPORTER GENES combinedneontological/ palaeontologicalapproach is the AND PROTEINS comparisonof acid-hydrolysis resistant, auto£uorescent Physiologicalstudies byR enault et al.(1992)in the mature placentaeof Coleochaete orbicularis (the onlycharo- early-divergentmoss stronglyimplicate hexose phyceanknown to produce placental transfer-like cells) sugars(rather thansucrose) asthe majorcarbohydrate (¢gure 4) and those ofbryophytes (Kroken et al. 1996). species that is movedfrom gametophyte to sporophyte. Analogously,morphologyof the placentalinterface ofthe Becausethe intergenerationaljunction is devoidof extantpteridophyte Tmesipteris elongata wasused byF rey symplastic connections(plasmodesmata) ,cell membrane et al.(1997)to postulate the occurrence ofplacentae in hexosetransporter proteins areimplicated. Such trans- lignieri ,basedon fossils that wereoriginally porters arethought to facilitate di¡ usion and/ oractively publishedby T aylor& Taylor( 1993). cotransportsugars and protons; homologous proteins in Depositionof resistant polymersinto the wallsof ,fungi, protists, higherplants and higher placentalcells occurs duringlate development of zygotes aremembers ofamajorfamily of transmembrane facilita- (Coleochaete )orsporophytes (bryophytes) .Thesematerials tors (Marger &Saier1993) . arenot present (as judgedby absence of speci¢c wallauto- Itis postulatedthat ,once imported into £uorescence andlack of resistance tohigh-temperature zygotes/sporophytes,is transformed intostarch orlipid acidhydrolysis) in vegetative thalli or during early zygote storage,or used more immediatelyfor embryo and sporo- development,including the periodof presumed solute phytedevelopment and spore production. Both processes transport. Thusdeposition appears to be underregulatory actas sinks that wouldtend topromote increased control.The function of resistant wallcompounds is not apoplastictransport fromgametophytic sources. In provenbut could include shutting o¡ solute £owor higherplants, sugars not only serve asenergysources and protectionfrom microbial attack (Kroken et al. 1996; buildingblocks for structural elements such ascellulose Graham,L. E.1996).Wallingrowths of Coleochaete are butare also potent regulatory molecules. Sugarsfunction alsoheavily impregnated and consequently resist acid assignalling molecules that activateor repress genes hydrolysis.As a result, cell wallingrowths, which repre- involvedin cell cycleregulation, and sent aphysicalmanifestation of matrotrophy ,arereadily pigmentproduction, glyoxylate , respiration, visibleat the lightmicroscopic level, both in bright ¢ eld starch andsucrose synthesis anddegradation, (¢gure 4) and UV -V £uorescence excitation.This suggests metabolism, pathogendefence and wounding responses, that ifancestral charophyceans related to Coleochaete ö andsenescence (Ehness et al.1997;Koch1996; Graham, organismstransitional between charophyceans and I.A. 1996;T ruernit et al.1996).Forexample, earliest embryophytes öorearly embryophytes also a/b bindingprotein genes and rbcS genesare - possessed such features, asis likely,theyshould have left repressible, whereasthe geneencoding nitrate reductase1 resistant remains inthe formof distinctive microfossils. issugar-inducible.F urther, genesessential forbiosynthesis Thusit is possiblethat microfossils youngerthan those ofethylene are activated by sugars, and those forbrassi- presently knownmay yet be found that willbe informa- nolidesare repressed. Receptors arehypothesized to be tiveregarding the earliest events inthe historyof integralcell membrane hexosetransporter proteins embryophytesand their acquisitionof alternation of (Lalond et al. 1999). generations.W earecurrently cataloguinghigh- Plantsalso possess sugarsensors. Theseare molecules temperature, acid-hydrolysedfertile thalliof various that detect the presence ofimported sugarsand transduce Coleochaete species andplacentae of modern bryophytes the signallingresponses. Thesugar withthe hopethat theymay provide useful search images enzymehexokinase (HXK) (whichalso functions at an forpalaeontologists as they seek remains ofearliest earlystep ofcytoplasmic ) is oneexample of a embryophytesor fossil evidenceof matrotrophy. sugarsensor ,becausethe sugarresponses ofhigherplants Neontologicalapproaches include comparative analyses require onlythat imported sugaris phosphorylatedby ofputative nutrient transport frommaternal cells to this ( Jang et al.1997).Itis thoughtthat the zygotes of Coleochaete andbetween gametophytic and signallingHXK is spatiallyassociated with membrane sporophytictissues ofearly-divergent embryophytes. W e hexosetransporters (Smeekens &Rook1 997).HXK is havefocused on comparative study of the hexosetrans- highlyconserved from bacteria to and higher plants porter genefamily in charophyceans and early-divergent andanimals, and is thus likelyto occur in charophyceans embryophytes(described inmore detailbelow) .Parallel andbryophytes, and to have similar sensingfunctions in e¡orts couldbe made to trace evolutionarychange in these . aminoacid transporters (Fischer et al. 1998) and H+- Webeganour analysis of hexose transport bydeter- .Genetic analysisof the regulationand biosynth- miningthat atleast some charophyceanalgae and bryo- esis ofwall-bound resistant polymersthat characterize phytes(e.g. Coleochaete , Graham et al. 1994; the mature charophyceanand bryophyte placentae is also charophyceandesmid ,Lewitus &Kana1 994;

Phil. Trans.R. Soc.Lond. B (2000) 764L. K. E.Grahamand L. W.Wilcox Alternation ofgenerations and several Sphagnum species, P.Bilkey,unpublished Partialsequences ofsimilar geneswere also obtained data)are capable of importing and using exogenous fromthe ,the liverwort Conocephalum hexosesugars, as deduced by growth experiments. This andseveral charophycean green algae with the same wasnecessary becausea number ofgreen algae are primers, but ampli¢cation product was not consistently knownto be incapable of utilizing hexose sugars. F or obtainedfor liverworts and charophyceans. Evidence that example,all of the (chlorophycean) Chlorococcum species ourprimers amplifyportions of hexose transporter tested byParker et al.(1961)wereincapable of utilizing proteins includeshigh similarity in primary sequence sugarsfor growth. The model-system Chlamydo- dataand in deduced hydropathy pro¢ les ofcharophycean monas reinhardtii (alsochlorophycean) is likewiseunable to andbryophyte/ aminoacid sequences inferred fromour utilize exogenoussugars (Harris 1989).Forsberg( 1965) DNA sequences andthose ofhomologous regions of concludedthat axeniccultures of Chara werealso unable HUP1(hexoseuptake protein 1 ),and Arabidopis toutilize exogenoussugars; however ,Smith (1967) STP1 (sugartransporter protein1 ). obtainedevidence that translucens cantake up and Inconclusion, it appearsthat the use of(i) neontological metabolize 14C-labelledglucose. approachessuch asmolecular analysis of matrotrophy in Wefoundthat hexoseutilization by charophyceans and charophyceansand bryophytes, and (ii) combinedneonto- bryophytesincreased under conditions in which dissolved logical/palaeontologicalapproaches in which resistant inorganiccarbon (DI C)waslimiting to photosynthesis. morphologyprovides links between extant charophytes Thissuggests alikelyadaptive advantage of hexose- andbryophytes and microfossils, arelikely to illuminate transport expressionin vegetative cells ortissues. Wethen the evolutionaryorigin of the embryophyticlife cycle to a hypothesizedthat hexosetransporter geneswere brought degreepreviously thought impossible. Such studies provide underincreased regulation in derived charophyceans notonly insight into evolutionary mechanism, but also havingretained zygotes, such thathexose sugars can be haveimplications for understanding the e¡ects ofearliest movedfrom green cells ofthe parentalgeneration to zygote plantson Ordovician biogeochemistry and may be useful storages,even under conditions when D ICis notlimiting to indeducing how the complexregulatory systems operating vegetativegrowth. Coleochaete producesgreater numbers of inhigher plant reproductive development have been meiospores thanany other green alga lacking alternation of constructed overevolutionary time. generations,and we hypothesize that matrotrophyexplains this di¡erence. Wesuggestthat matrotrophywas inherited byembryophytes from ancestral charophyceans and used as Wearegrateful to Colleen Lavin and the Integrated Micro - scopyResource at the University of Wisconsin-Madison for ameans ofsupportingampli¢ ed spore production, and in assistancewith high-pressurefreezing techniques. John Raven aspects ofsporophytemorphogenesis. andDianne Edwards very kindly reviewed the manuscript and Becauserapid diversi¢ cation of -speci¢ c members o¡ered helpful suggestions. W ealsoacknowledge N ational ofgene families is involvedin the originof functional ScienceF oundation(USA) grant DEB 9628869, our colleagues innovationsand evolutionary radiation in other - MadelineFisher, Evan Lau and Robin Kodner for assistance ismal groups(Iwabe et al.1996;Heniko¡ et al. 1997), we with thehexose transporter sequencing work, and Brent areexploring the possibilitythat geneduplication, diver- Mishlerand Louis Lewis for providing moss and liverwort DNA. genceand tissue-speci¢ c expressionplay important roles inthe evolutionaryorigin of plant matrotrophy .Weare currently testing these hypothesesby cataloguing sequences ofhexose-transporter genefamily members in REFERENCES selected charophyceansand bryophytes, then usingthe Bai,X., Peirson, B. N.,Dong, F .,Xue,C. &Makaro¡,C. 1999 informationto study expression patterns. Wepostulate Isolationand characterization of SYN1, a RAD-like gene that speci¢c genefamily members mayoccur only in essentialfor meiosis in Arabidopsis . 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Thehypothesis may reasonably be extendedto Ulvales, otherin a bigocean, but few studies havefocused on thoughintimate associationof sporophytes and gameto- matingsuccess rates inalgae. Such data could be helpful phytesis lacking.Multicellular ulvalean sporophytes can intesting the hypothesis. clearlyproduce greater numbers ofmeiospores thancan presumed ancestralforms (modelledby Ulotrichales Reference whosediploid stage is unicellular),andthis couldbe Hommersand,M. S. &Fredericq,S. 1990Sexual reproduction advantageousin the marinehabitat (Graham &Wilcox andcystocarp development. In Biologyof the red algae (ed. 2000).Itis possiblethat matingfrequency in ulvaleans K.M. Cole& R.G.Sheath) ,pp.305 ^346.Cambridge couldbe limited bythe abilityof gametes tolocate each UniversityPress.

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