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University of Chicago Press Pollen Wall Development of Austrobaileya maculata Author(s): Michael S. Zavada Source: Botanical Gazette, Vol. 145, No. 1 (Mar., 1984), pp. 11-21 Published by: University of Chicago Press Stable URL: http://www.jstor.org/stable/2474507 Accessed: 23-10-2015 14:38 UTC

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BOT. GAZ. 145(1):11-21. 1984. ? 1984 by The Universityof Chicago. All rightsreserved. 0006-8071/84/4501-00 11$02.00

POLLEN WALL DEVELOPMENT

OF AUSTROBAILEYA MACULATA

MICHAEL S. ZAVADA Departmentof Biology,Indiana University, Bloomington,Indiana 47405 Pollen wall development of Austrobaileya maculata was investigatedfromthe initiationof pollen mothercells to anthesis. Pollen wall developmentcan be divided into threephases: premeiotic,tetrad, and freespore. The ektexinedevelops withinthe tetradphase and followsthe patternforothertectatecolumellate angiosperm pollen thus far investigated. The endexine develops in the free-sporephase and has two modes of deposition, depending on whether it is apertural or nonapertural. Intine developmentbegins priorto the completionof the endexine and becomes interlayeredwith the endexine, causing the endexine to corrodewhen acetolyzed because of the solubilityof the intine. The last major developmentalevent is the injectionof tapetal substances intothe intersticesof the ektexine.At anthesis the pollen is binucleate and has fullydeveloped ektexine (with tapetal substances occupying its interstices),endexine, and intine.

Introduction Austrobaileyaceaeincludes one genus and two species(Austrobaileyamaculata and A. scandens) distributed in tropicalrainforestsofNorthQueensland, Australia. The primitivenatureof Austrobaileya has attractedconsiderableattention,and the genus is consideredimportantto our understandingof angiospermphylogeny(BAILEY and SWAMY 1949; ENDRESS 1980). Wall ultrastructure of maturepollen grainswas studiedby WALKER (1976) and ENDRESS and HONEGGER (1980). The pollenis monosulcateand reticulate,and the wall structureis tectatecolumellatewith a thin footlayer.The endexineis a thin,continuousgranular layerin nonaperturalregionsand is thickand lamellatedin aperturalregions.Pollen wall developmentof A. maculate was studiedfromthe initiationofthepollenmothercells(PMCs) to anthesis and, on the basis of major developmentalevents, can be divided into threephases: premeiotic,tetrad, and freespore.

and embedded in Dow Epoxy Resin-334(DER334). Thin sectionswere cut on an LKB-1 ultramicrotome, stainedfor15 minwithaqueous uranyl acetate and lead citrateor with KMnO4 and lead citrate,and examinedon a Philips EM-300 at 80 kV. Acetolyzedpollen (ERDTMAN1969) was prein an alcoholseries, paredforTEM bydehydration embeddingin DER-334, and sectioningand staining as above. SCANNING ELECTRON MICROSCOPY (SEM)

Antherswereeitherfixedas forTEM, or pollen was acetolyzed(ERDTMAN1969),mountedon stubs withthehigh-vacuumwax ApiezonW-100,coated withgold palladium,and viewed on a Coates and Welterfieldemissionscanningelectronmicroscope.

Observations AustrobaileyamacuBuds of greenhouse-grown lata are initiatedin late December (late springhemisphere). Buds 1-6 mm earlysummer,southern in diameterhave antherswith pollen in the premeioticphase and average81 daysto anthesis.The Material and methods anthertissuein thesebuds exhibitedverylittledeTRANSMISSIONELECTRON MICROSCOPY(TEM) velopmentalactivityshortlyaftertheirinitiation (lateDecemberto late February,latesummer-early Anthers of greenhouse-grown Austrobaileya maculata(voucherdata: collector,Zavada; collec- fall, southernhemisphere).Buds 7-11 mm in diameterhave antherswithpollenin thetetradphase tionno. 504; Herbarium,CONN) weresampledat All budswere and average47 daysto anthesis.Buds 12 to greater variousintervals duringdevelopment. measuredand taggedto determinetherelationship than 18 mm in diameterhave pollen in the freesporephase and average5 days to anthesis.Flowbetweenbud size and the developmentalphase of eringis completedby mid-lateMay. No fruitsdethepollenand to determinethenumberof days to veloped, suggesting that A. maculata is selfanthesis.Antherswere fixedin 3% 0.1 M cacoincompatible. dylateHCl-buffered glutaraldehyde-formaldehyde inan ice bathfor1-8 h, washedin cacodylatebuffer, PREMEIOTIC PHASE and postfixedin 2% aqueous Os04 for 1-4 h at Priorto PMC formation,the sporogenouscells 0 C. Tissues were dehydratedin an alcohol series are characterizedby thinprimarycells walls with profile(fig. plasmodesmata and a normalcytological Manuscript received November 1982; revised manuscript 1). Presentare many of the organellesfound in receivedAugust 1983. 11


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ZAVADA-AUSTROBAILEVA

parenchymatous tissuesofotherplantorgans.The PMC formation is initiatedin thecentralregionof theantherloculeand proceedscentrifugally. As the formationof the PMCs continues,plastidsaccumulatelargedepositsofstarch,and thePMC plasmalemmapulls away fromthe primarycell wall, severingplasmodesmatalconnections(figs.2, 11). The primarycell wall thenlooselysurroundsthe PMC and appears to be partiallydigested;however,thiswall is persistentand will adhereto the newlydevelopingcallose special wall (CSW) (fig. thePMC protoplastbecomescir3). Concurrently, cularin outline,and callose is formedby the dischargeofthecontentsofGolgi-derived vesiclesinto the area between the CSW and the PMC plasmalemma(fig.3). At thistimethenucleusappears to enlarge,markingthe beginningof meioticprophase. Eventually,cytokinesis occurs,and thecell walls ofthetetradare formedcentripetally (fig.4). Callose depositioncontinuesuntilall fourmicrosporesare surroundedby the CSW, signalingthe beginningof the tetradphase (fig.5). Afterthe most peripheralof the PMCs have formed,the tapetumbecomes a recognizableunior bilayeredtissue (fig. 11). Developmentof the tapetalcells proceedsby the plasmalemmapulling awayfromtheprimarycellwall and bysubsequent digestionof theprimarywall. Duringthelate premeiotic,tetrad,and free-spore phases,thetapetum is secretary. TETRAD PHASE

Afterthefourmicrospores have beensequestered by the CSW, an electron-dense fibrillarwall, the is depositedbetweentheplasmalemma primexine, and theCSW (figs.6-10). Fromtheonsetofprimexineformation, less dense areas radiallytraverse themoreelectron-dense fibrillar primexine(fig.6). Beneaththeseless denseareas are vesiclesobliquely orientedin the cytoplasm(figs.6, 7). These are In tangentialsecsitesofprocolumellaeformation. tionsof the primexine,the vesiclesthat are associatedwiththeless dense areas of primexineoften appear multivesiculate (fig.8). Afterprimexineformation is completed,circular membraneprofilesare evident beneath the procolumellae,and the vesicles associated with procolumellaeformation disappear(fig.9). Soon after circularmembraneprofilesappear, procolumellae becomemoreelectrondense distallyand centripthe circularmemetally(fig. 10). Concurrently, brane profilesbecome less evident(fig. 10), and lateralaccretionofprotosporopollenin occursat the of the distal ends of the columellae;construction tectumensues(fig.10). Followingtheformation of theprotosporopolleninous tectumand columellae, nexine1 (footlayer) accretionbegins. continuesthroughout thereFootlayerformation mainderof the tetradphase and is completedby

MACULATA

13

timetheCSW is destroyedaroundthemicrospores. The sporopolleninin the footlayeris accumulated on long,unit-likemembranesthatbecomesequentiallyappressedand fusedto the bases of the coloftheektexine(fig. umellae,completingformation memtheunit-like formation, 12). Duringfootlayer branesare recognizablebut becomeless evidentas formation nearscompletion.The position footlayer even oftheunit-likemembranescan be visualized,afterthe footlayerappears homogeneous,by immersingthe thinsectionsin acetolysissolutionfor 30-60 s, followedbyuranylacetateand lead citrate has been fully staining(fig.13). Afterthefootlayer formed,theCSW is destroyedand themicrospores are released into the antherlocule with fullydeveloped tectum,columellae,and footlayer. The positionof the sulcus in Austrobaileyais determinedearlyin thetetradphase by thefailure of theprimexineto developin thisregion(fig.14). In the regionsadjacent to the aperture,the primexine taperssomewhatand eventuallydisappears in the area of the futureaperture(fig.14). Duringthetetradphase,thetapetalcellsare unior binucleateand becomemoreactive.The tapetal byabundantsmoothencytoplasmis characterized doplasmicreticulumand plastidswithstarchgranules. The most conspicuousevent in the tapetal cells duringthis phase is the formationof sporoof thelocular polleninousplaques in theproximity plasmalemma(fig.29). The granularplaques will eventuallydevelop into orbicules (figs. 30, 31), which remain in proximityto the plasmalemma the tetradphase and are releasedinto throughout the antherlocule afterdissolutionof the CSW at phase. the beginningof the free-spore FREE-SPORE PHASE

Afterdissolutionof the CSW, the microspores expand ca. 2-3 timestheirpreviousdiameter.At thesame time,formation ofthe granularnonaperturalendexinebegins.The endexineis formedby the apposition of sporopolleninousgranules exand the trudedintothearea betweenthefootlayer microsporeplasmalemmaby vesicles(figs.15, 17, 18). Even when sectionsof Austrobaileyaare exposed to acetolysis,white lines (the positionformallyoccupiedby theunitmembranes)can be observed in the footlayer;however,the granulesof the endexinein the nonaperturalregionsremain homogeneous(fig.16). The endexineis thickestin theaperturalregion. In contrastto the nonaperturalendexine,it is accretedon unit-likemembranes,resultingin a lamellatedappearance at maturity(fig.22). In addition,considerableamountsofintineinterdigitate endexine.Thus, withthesheetsof sporopolleninous to determine whenendexineformation itis difficult ends and intineformation begins(fig.19). The inof the endexineand intinein apertural terlayering


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Fig. 3, PMC with numerous plastids and thick callose special wall (CS W) surrounded by the remnantsof FIGS. 3-8. the primarycell wall (PCW). Note Golgi derived vesicles (GDV) and plasmalemma. x 7,800. Figs. 4-8, The tetrad phase. Fig. 4, Meiotic cytokinesisshowing centripetalformationof the microsporecell wall (CW, directionof arrows). x 17,000. Fig. 5, Tetrahedral tetrad surrounded by callose. x 1,000. Fig. 6, Formation of the primexine (PX); note the obliquely orientedvesicles (arrow) in the less dense areas where the procolumellae form. x 33,800. Fig. 7, Oblique section through the developing primexine,showing "vesicular" extensions of the plasmalemma underlain by tubular structures(arrows). x 33,800. Fig. 8, Tangential section of the developing primexine(PX) showing circular membrane profilestraversingthe primexine.Also note "finger-like"extensionof the plasmalemma (arrow). x 33,800.


ZAVADA-AUSTROBAILEYA

and nonaperturalregionsmay accountforthetendency of the endexineto corrodeupon acetolysis ofmaturepollengrains.The intine,whichpartially consolidatesthe endexinal layer, is dissolved on treatment withacetolysissolution,fragmenting the endexine(comparefigs.27, 28). As soon as the intinebecomes distinguishable, theektexinebeginsto lose affinity fortheEM stains uranylacetateand lead citrate;however,endexine

MACULATA

15

maintainsits dark-staining propertieseven at maturity(figs.19-21). During the free-sporephase, the tapetumdischargesorbiculesand othercytoplasmicdebrisinto the antherlocule (e.g., membranes;figs.30, 31). Duringthelate free-spore phase, largeamountsof tapetal substancesare depositedin the interstices of the pollenwall (figs.23-26). Thereuponthe tapetum begins to lose its integrity and eventually

FIGS. 9-13. -Tetrad phase. Fig. 9, Procolumellae formation;procolumellae are oftenassociated with the circular membrane profilesbetween the plasmalemma and primexine(arrow). At this stage the tubular structuresassociated with the areas of procolumellae formationare no longer evident. x 43,700. Fig. 10, Procolumellae traversingthe primexine;the circularmembraneprofiles,presentearlier,are no longerevident. Shortlyafterthe circularmembraneprofilesare no longer evident,the distal ends of the procolumellaebecome electrondense (arrow) x 30,700 Fig 11, Premeioticphase. Formation of pollen mothercell (PMC) and tapetum (TC). PCW, primarycell wall. x 14,300. Fig. 12, Footlayer formationquickly ensues afterthe columellae and tectumbecome distinct.Footlayer is laid down on unit-likemembranes. x 4,500. Fig. 13, Acetolyzed section showing lamellations in the footlayer(arrows). x 17,600.


16

BOTANICAL GAZETTE

pollen. The developmentof the sporopolleninous ektexinecommencesafterthefourmicrospores have been sequesteredby the CSW (tetradphase) and formationof the primexineis completed.An interestingaspect of primexineformationis the associationof vesicularstructuresin the peripheral Discussion cytoplasmwiththeprimexinalareas at thesitesof Pollenwalldevelopment ofAustrobaileyais sim- the procolumellae.Thus far,thesevesicularstrucilartothatinothertectate-columellate angiosperm tureshave been observed only in Zea (SKVARLA breaks down, signalingthe onset of anthesis(fig. 32). At anthesisthe pollen grains are binucleate and have fullydeveloped ektexine,granularnonaperturalendexine,lamellatedaperturalendexine, and intine.

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x 10,60 Fis 15,16, Te fre-spoe phae Fig15, fter issoltion f th callse spcial allendexne (E) foratio ensuesbythe appositionof granules,whichcan be interbedded withlargergranulesappearingto be formedon unit like~~~~~~~~~~~~~~

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pollenshowingreticulate FIGS. 23-28.-Free-spore phase. Figs. 23-26, SEM. Figs. 27. 28, TEM. Fig. 23, Acetolyzed sculpturing. x 2,700. Fig. 24, Nonacetolyzed pollen showing numerous tapetal substances deposited in and on the pollen wall. x 2,700. Fig. 25, Acetolyzedpollen showingreticulateexine sculpturingand the undulatingappearance of the tectum, an artifactof acetolysis. x 7,500. Fig. 26, Nonacetolyzed pollen showingthe substances deposited in the tectal perforations and the smooth nature of the tectum. x 7,500. Fig. 27, Acetolyzedpollen showing the lack of an endexine which has been destroyedby acetolysis. x 21,000. Fig. 28, Nonacetolyzed pollen showing homogeneousendexine (EN) and the somewhat electron-densetapetal substances (TS) deposited in the intersticesof the wall. x( 21,000.



and LARSON 1966). The mechanismthat determinesthe positionof the columellaeand the role these vesicles play in this regard are unknown. However,theassociationof membraneprofiles with columellaeformationseemsto be commonamong angiosperms(DICKINSON and HESLOP-HARRISON 1968). The positionof the sulcus is also determinedin thetetradphase and resultsfromthefailureofthe primexineto develop in this region.The mode of aperturedetermination is similarto thatin Silene (HESLOP-HARRISON 1963a, 1963b), Helleborus (ECHLIN and GODWIN 1968),Beta (HOEFORT 1969),

MACULATA

19

Citrus(HORNERand LERSTEN 1971), and Capsicum(HORNERand ROGERS1974).

The mode of endexineformationin Austrobai-

(ECHLIN and leyais similarto that in Helloborus GODWIN1969),Passifiora(LARSON1966),and Zea

and LARSON1966).In otherangiosperms (SKVARLA HORNERand PEARSON1978; Silene, (Helianthus, SHOUPet al. 1980), endexineis accretedon unitlike membranes,impartinga lamellated appearance to maturenonaperturalendexine. DOYLE et al. (1975) discussedthe importanceof fossilangiospermouspolendexineforidentifying len. They determinedthat mature,nonapertural

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FIGS. 29-32.-Development of the tapetum. Fig. 29, Secretorytapetum during the late tetrad phase with numerous sporopolleninousplaques (arrows) on the tapetal cell plasmalemma. x 3,030. Fig. 30, Tapetal cells duringearly free-spore phase and the discharge of the electron-denseorbicules (0) into the anther locule. x 4,800. Fig. 31, Orbicules (0) being discharged into the anther locule during the mid-free-sporephase. Note ektexine(EK) and orbicules (0) are beginningto lose theiraffinityforthe EM stains uranyl acetate-lead citrate. x 5,000. Fig. 32, Late free-sporephase; orbicules (0) and ektexine(EK) have lost their affinityfor the EM stains uranyl acetate-lead citrate; tapetum is beginningto break down. x 5,000.


20

BOTANICAL GAZETTE

[MARCH

endexinein angiospermsis nonlamellatedat maturity;in contrast,the"endexine"of gymnosperms is lamellated in nonaperturalareas at maturity. However, the lamellated basal layer of gymnospermsis homologousto the footlayerof angiosperms(nexine1 of ERDTMAN1969),not the endexine (nexine 2 of ERDTMAN 1969). The basal layerof gymnosperms developsentirelyin the tetrad phase, in the same way thatthe footlayerdevelops in angiosperms(on long, unit-likemem-

bilitysystemsand can constitute18% oftheweight ofa singlepollengrain(GILLISSEN and BRANTJES phaseofthetapetum 1978).The extensivesecretary in angiospermsappears to be an importantand conspicuous developmental event. Although have a similarsecretaryphase, it is gymnosperms not as extensive,and the amount of tapetal substancesdepositedin or on thepollenwall is minute comparedwith angiosperms(Zamia, ZAVADA,in preparation). Austrobaileyais considereda morphologically branes; VASIL and ALDRIDGE 1970; DICKINSON 1971; WILLEMSE 1971; AUDRAN 1981; ZAVADA,in primitiveplant. However, aside fromhavingthe primitiveaperturalcondition,a monosulcus,it has preparation).In addition,some angiospermshave lamellatedfootlayers at maturity (Annonaceae,LE featuresconsideredderivedin angiospermpollen, and endthemorphological and wall structure i.e., thetectate-columellate THOMAS1981),underscoring developmentalsimilarityof gymnospermnexine exine(WALKER1976). Its pollenwall development (nexine1, sensu stricto)and the angiospermfoot- exhibitsno deviationfromthe patternof wall delayer.No othersporopolleninous wall layerdevel- velopmentobservedin otherangiospermsnor any ops duringthe free-spore phase in gymnosperms. similaritiesor peculiaritiesof pollen wall develwhich can supportits Therefore,thereis no wall layerdevelopmentally opmentin gymnosperms, equivalentto the angiospermendexinein gymno- primitivestatus. Thus, the pollen characteristics wall sperms,and theendexineappearsto be foundonly observedin Austrobaileya-tectate-columellate in angiosperms. structure, presenceofan endexine,and pollenwall The interlayering of the endexineand theintine developmentsimilar to other tectate-columellate also occurs in Helleborus(ECHLIN and GODWIN angiospermpollen-are all characteristics of more 1969) and Passifiora(LARSON1966). In acetolyzed advancedangiosperm pollenand emphasizethehigh of Austrobaileyapollen, the endexineis fragmented level of mosaic evolutionthat is characteristic and appears scantyor absentbecause of the solu- thefamilyAustrobaileyaceae in particular and many and bilityoftheintinematerialthatis interlayered familiesof primitivedicotyledonsin general. partiallyconsolidatesthe endexine(figs.27, 28). This mustbe kept in mindwhen interpreting the Acknowledgments natureand extentof endexinein fossiland extant acetolyzedpollen. I thank Drs. WILLIAM L. CREPET, University The last major developmentaleventof thefree- of Connecticut, THOMAS N. TAYLOR, Ohio State spore phase is the injectionof tapetal substances University, JAMES WALKER, University of Mas(pollenkitt)into the intersticesof the pollen wall. sachusetts,and an anonymousreviewerfortheir This is concomitantwiththefinaldevelopmentof manyhelpfulcomments.This projectwas partially the intine.These tapetal substances,in manyansupportedby National Science Foundationgrant giosperms,play a role in intraspecific incompati- 8110217 to W. L. CREPET. LITERATURE J. C. 1981. Pollen and tapetum development in Ceratozamia mexicana (Cycadaceae): sporal origin of exinic sporopollen in in cycads. Rev. Paleobot. Palynology 33:315-346. BAILEY, I. W., and B. G. L. SWAMY. 1949. The morphology and relationships of Austrobaileya. J. Arnold Arboretum 30:211-226. DICKINSON, H. G. 1971. The role played by sporopollenin in the development of pollen in Pinus banksiana. Pages 31-67 in J. BROOKS, P. R. GRANT, M. MUIR, P. VAN GIJZEL, and G. SHAW, eds. Sporopollenin. Academic Press, London. DICKINSON, H. G., and J. HESLOP-HARRISON. 1968. Common mode of deposition forthe sporopollenin of sexine and nexine. Nature 220:926-927. DOYLE, J. A., M. VAN CAMPO, and B. LUGARDON. 1975. Observations on exine structure of Eucommiidites and Lower Cretaceous angiosperm pollen. Pollen Spores 12:429-486. ECHLIN, P., and H. GODWIN. 1968. The ultrastructure and ontogeny of pollen in Helleborus foetidus L. II. Pollen grain development through the callose special wall stage. J. Cell Sci. 3:175-186. AUDRAN,

CITED . 1969. The ultrastructure and ontogeny of pollen in Helloborusfoetidus L. III. The formation of the pollen grain wall. J. Cell Sci. 5:459-477. ENDRESS, P. K. 1980. The reproductive structures and systematic position of the Austrobaileyaceae. Bot. Jahrb. Syst. 101:393-433. P. K., and R. HONEGGER. ENDRESS, 1980. The pollen of the Austrobaileyaceae and its phylogenetic significance. Grana 19:177-182. ERDTMAN, G. 1969. Handbook of palynology. Hafner, New York. L. J. W., and N. B. M. BRANTJES. 1978. Function GILLISSEN, of the pollen coat in differentstages of the fertilization process. Acta Bot. Neerlandica 27:205-212. HESLOP-HARRISON, J. 1963a. Ultrastructural aspects of differentiation in sporogenous tissue. Symp. Soc. Exp. Biol. 17:315340. . 1963b. An ultrastructural study of pollen wall ontogeny in Silene pendula. Grana 4:1-24. HOEFORT, L. L. 1969. Ultrastructure of Beta pollen. I. Cyto-


1984]


plasmic constituents. Amer. J. Bot. 56:363-368. HORNER, H. T., and N. R. LERSTEN. 1971. Microsporogenesis in Citrus limon (Rutaceae). Amer. J. Bot. 58:72-79. HORNER, H. T., and C. B. PEARSON. 1978. Pollen wall and aperture development in Helianthus annuus (Compositae: Heliantheae). Amer. J. Bot. 65:293-309. HORNER, H. T., and M. A. ROGERS. 1974. A comparative light and electron microscopic study of microsporogenesis in male fertile and cytoplasmic male sterile pepper (Capsicum annuum). Can. J. Bot. 52:435-441. LARSON, D. A. 1966. On the significance of the detailed structure of Passiflora caerulea exines. BOT. GAZ. 127:139-154. LE THOMAS, A. 1981. Ultrastructural characters of the pollen grains of African Annonaceae and their significance for the phylogeny of primitive angiosperms. Pollen Spores 22:267342. SHOUP, J. R., J. OVERTON, and M. RUDDAT. 1980. Ultrastructure and development of the sexine in the pollen wall of Silene

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alba (Caryophyllaceae). BOT. GAZ. 141:379-388. SKVARLA,J. J., and D. A. LARSON. 1966. Fine structureof Zea mays pollen. I. Cell membranes and exine ontogeny. Amer. J. Bot. 53:1112-1125. VASIL, I. K., and H. C. ALDRIDGE. 1970. A histochemical and ultrastructural study of the ontogeny and differentiationof pollen in Podocarpus macrophyllusD. Don. Protoplasma 71:137. WALKER, J. W. 1976. Evolutionary significance of the exine in the pollen of primitive angiosperms. Pages 251-307 in I. K. FERGUSON and J. MULLER, eds. The evolutionary significance of the exine. Academic Press, London. WILLEMSE, M. T. M. 1971. Morphological and fluorescence microscopical investigation on sporopollen formationof Pinus sylvestrisand Gasteria verrucosa. Pages 68-107 in J. BROOKS, P. R. GRANT, M. MUIR, P. VAN GIJZEL, and G. SHAW, eds. Sporopollenin. Academic Press, London.
