Apical Cell Segmentation and Its Relationship to the Peristome-Forming Layers in the Author(s): Owen M. Schwartz Source: International Journal of Sciences, Vol. 158, No. 3 (May, 1997), pp. 236-248 Published by: The University of Chicago Press Stable URL: http://www.jstor.org/stable/2475273 Accessed: 17/11/2010 10:28

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http://www.jstor.org Int. J. Plant Sci. 158(3):236-248. 1997. ? 1997 by The Universityof Chicago. All rightsreserved. 1058-5893/97/5803-0002$03.00

APICALCELL SEGMENTATION ANDITS RELATIONSHIP TOTHE PERISTOME-FORMINGLAYERS IN THEFUNARIACEAE

OWEN M. SCHWARTZ' Departmentof Botany,Duke University,Durham, North Carolina 27706

Six taxa in the Funariaceae were examined to compare developmentand cell division patternsin capsules with and withoutperistome teeth. The peristomedifferentiates from a series of apical cell segmentsthat are foundto develop by a similar patternof cell divisions. The youngestapical cell segmentsdo not complete the entiredevelopmental sequence observed in the more proximalsegments. Few differencesin developmentwere foundamong the taxa, other than in the timingof transversecell divisions. Segmentsin the gymnostomoustaxa (Ephemerum,) do not complete the entire sequence of divisions, with only three amphitheciallayers present in the mature capsule. Spiralingof the peristometeeth of is suggestedto be the resultof differentialcell expansionduring the course of developmentof the distal apical cell segments,although oblique division of the apical cell mightalso be responsible for the spiralingof the cell files. Moss sporophytesare bilaterallysymmetrical in theirdevelopment although forming a radially symmetricalcapsule. Because peristometeeth differentiate from several differentlevels in several different segments,cell position as well as cell lineage obviously is responsiblefor guidingdifferentiation. Introduction these apical cell derivativeshave been describedmany times,although not withrespect to peristometooth for- Although aspects of peristomedevelopment in the mation (De Bary 1884; Kienitz-Gerloff1878; Sachs have been studied for more than a century 1887; Goebel 1905; Evans and Hooker 1913). Some (Sachs 1887; Goebel 1905; Campbell 1913; Smith differencesin the orientationof early divisions in the 1955; Proskauer1958; Shaw et al. 1987, 1989), apical segmentsof Funaria and Ephemerumwere described segmentationand its relationshipto peristomedevel- but were not traced to the final morphologyof the opment have not been well documented.Aside from capsule. studies on embryology,nearly all published illustra- The sequence of cell divisions withina given seg- tions are of cross sections of sporophytes,probably ment of certainother taxa has been described as ex- because the importantsystematic characters associated tremelyregular. In the shoot apices of liverworts,a with the peristomeare usually seen in this plane of precise series of cell divisions within each segment section. In particular,development as viewed in three gives rise to leaves and lateral branches in specific dimensions has never been fully explored. A three- positions (Schuster 1966). Precise patternsof cell di- dimensionalview of developmentreveals the relation- vision withinsegments in the roots of heterosporous ship between cell lineages, segments sensu Gifford ferns,e.g., Azolla, also show thatcell position within (1983), Gunning(1978), and Schuster(1966), and dif- a segmentguided differentiation(Chiang 1970; Gun- ferentiation. ning et al. 1978). In contrast,initiation of leaf primor- A single meristematicinitial is presentat the apex dia in fern apices depends only on position on the of the Funaria sporophyte(Hofmeister 1862). Deriv- apex, withno regardto segmentboundaries (Bierhorst atives of the apical cell, oftenreferred to as segments, 1977). Are segmentboundaries and cell positionwith- are formedby inclinedtransverse divisions on two cut- in a given segmentimportant in guidingdifferentiation two vertical tingfaces, and derivativesare arrangedin of peristometeeth? Regular patternsof cell division files on opposite sides of the sporophyteaxis. Early in and leaf initiationwithin segmentsof moss gameto- the cell divides sporophytegrowth, apical frequently phyteshave been reported(Merl 1917; Berthier1971- (Hebant 1973), cuttingoff 10-14 segments.From two 72), but segmentationin moss sporophyteshas not of the earliestapical cell segments,an intercalarymer- been completelyinvestigated. The lack of information istem differentiates(Roth 1969; French and Paolillo led Proskauer(1958) to point out thatthe relationship 1975, 1976). Elongation of the intercalarymeristem of the peristomiallayers to apical cell segmentation derivativesis responsiblefor the increase in lengthof had never been properlydescribed. the seta portionof the sporophyte.Segments formed I initiatedthis investigationto resolve differingde- afterthose differentiatinginto the intercalarymeristem in the literatureand to relate apical cell seg- remain,with few internaldivisions, until much laterin scriptions development. mentationto peristometooth differentiation. Although of was re- The firstthree or fourplanes of cell division within the developmentof the peristome Funaria cently reinvestigated(Shaw et al. 1989), apical cell segmentationwas not considered. Patternsof cell di- 'Address for correspondence and reprints:Plant Cell Biology Group,Research School of Biological Sciences, GPO Box 475, Aus- vision and apical cell segmentationin cleistocarpous tralianNational University,Canberra, ACT 2601 Australia;E-mail: taxa are even less well understood,with only a very [email protected]. limited number of taxa examined (Hofmeister1862; Manuscript received July 1996; revised manuscript received Muller 1868; Kienitz-Gerloff1878; Sachs 1887; Goe- December 1996. bel 1905; Snider 1975; Rushing and Snider 1980).

236 SCHWARTZ-FUNARIA APICAL SEGMENTATION 237

None of these studies described segmentationduring the later stages of development. 3 The following questions will be addressed: Do all A apical cell derivativesfollow the same patternof de- velopment?Do taxa with peristomesfollow the same patternof developmentas cleistocarpous taxa? How ' , 1 ~~~~~~~7b are the positionsof the peristometeeth related to seg- 7a ~ 7b mentboundaries? Materialand methods Moss sporophytesand attachedgametophytes were fixed in FAA (formalin-aceticacid-ethanol 1: 1:18). Afterfixa- 14~~~~~~~~1 tionwas complete,sporophytes and embryos were dissected fromthe archegonia. were cleared in a diluteClorox solution,dehydrated through a gradedethanol series, and stainedin fastgreen FCE Whole embryosand sporophyte 1 4~~~~ 11 1W 8 apices weremounted in gumdammar or cedaroil and pho- tographedon a LeitzOrtholux microscope. For complete de- Fig. 1 Schematic diagram of cell divisions withina single seg- tailsof themethods used, see Schwartz(1994). ment of Funaria. The outer surfaceof the sporophyteis to the left In orderto followthe cell divisionpattern within a single on each segment.The rightside of each segmentforms the central segment,it is essentialto comparethe development of sev- axis of the sporophyte.The numbersadjacent to each segmentare erallayers of cells.Therefore, cleared sporophyte apices and cell division planes which formthe common developmentalpattern embryoswere used ratherthan longitudinal sections. This followed by all segments.The apparentgaps in the numberingsys- tem are anticlinal divisions which cannot be seen in longitudinal permittedoptical sectioning of thesporophytes and compar- section.A, An undividedsegment. The firstdivision of the segment ison of surfacewith median views within the same sample. cannot be seen in longitudinalsection because it is parallel to the Furthermore,segment boundary walls are muchmore dis- plane of section, as are all anticlinal/radialdivisions. 2 = the first tinctin clearedsporophytes than in longitudinalsections. If periclinaldivision; 3 = the firsttransverse division; 5 = formation longitudinalsections were used, segmentboundary infor- of the second amphitheciallayer; 7a = transversedivisions of the mationwould be lost. lower half of the segment; 7b = transversedivisions of the upper half of the segment;8 = formationof the thirdamphithecial layer; Definitions 11 = formationof the fourthamphithecial layer; 13 = the third series of transversedivision planes (a-d are the transversedivision Apical cell segments,or merophytes,are immediate planes in each 1/4 segment); 14 = formationof the fifthamphithe- unicellularderivatives of the apical cell. Because there cial layer; 16 = transversedivisions of the PPL; 18 = formationof are two cuttingfaces of the apical cell, thereare two the sixth amphitheciallayer. verticalfiles of segmentson opposite sides of the spo- rophyte.The segmentsare numberedin the sequence in which theywere cut offfrom the apical cell. There- The division planes are numberedrather than the in- fore,segment 1 is the mostrecently formed, while seg- dividual cell divisions. ment 14 is the oldest in the case of Funaria, for ex- Anticlinaland periclinalcell divisionplanes will not ample. be emphasized here, since theyhave been treatedpre- Three layersof cells are involved in peristometooth viously (Schwartz 1994). Rather,transverse divisions, formationin Funaria. The exostome teeth,the outer not visible in cross section,and detailsof segmentation ring of peristometeeth, are formedby thickeningson will be discussed. the common wall of the outerperistomial layer (OPL) and the primaryperistomial layer (PPL). The endo- Results stome or inner are formedfrom segments, peristome, DEVELOPMENT OF FUNARIA the common wall of the primaryperistomial layer (PPL) and the innerperistomial layer (IPL). The IPL In embryos200 ,umlong, six to eight segmentsare layer is the innermostlayer of the amphithecium.A present.In embryosof about 4 mm,the apical cell still morecomplete description of these layerscan be found cuts offderivatives, and thin-walled,undivided, newly in Schwartz (1994) and Shaw and Robinson (1984). initiatedsegments are parallel and proximalto the cut- Throughoutthe descriptivesections, reference will tingfaces of the apical cell (fig. 2A, B, E). The apical be made to particularnumbered planes of cell division. cell ceases to divide aftercutting off a total of ap- These numberswill appear throughoutthe text in bold- proximately14 segments,the earliest of which give face, referringto Funaria, the taxon with the most rise to the seta and the laterof which formthe capsule complex development.The numbersrefer to the line and peristome.As capsule maturityapproaches, divi- drawingsof Funaria segments(fig. 1), althoughfigure sions and differentiationbegin to take place in the 1 will not be specificallyindicated with each division youngersegments near the apical cell, which untilthis plane number.Each division plane representsmany point remained distinct,with few internaldivisions. separate cell divisions, all in the same plane and par- The overall developmentalsequence is of alternating allel planes, occurringat approximatelythe same time. anticlinal,periclinal, and transversedivisions. 238 INTERNATIONAL JOURNAL OF PLANT SCIENCES

A* _ B f~~~~~i AlC

S1~~~~~~~~~~~~~~~~~

B' F SCHWARTZ-FUNARIA APICAL SEGMENTATION 239

DEVELOPMENT WITHIN A SINGLE SEGMENT AT only in the second ringof amphithecialtissue, the PPL THE LEVEL OF THE ANNULUS (fig. 3E). Each cell of this verticalfile is divided into The initial anticlinaldivision (1) is difficultto see, basiscopic and acroscopic cells. At the conclusion of but it is visible in optical section(Al in fig.2C). When this round of divisions there are twice as many cells the two youngestsegments have completed theirfirst in this vertical file as there are in the IPL, or OPL, divisions,four quadrants are formed,two in each seg- which are immediatelyadjacent. At the time of ma- ment.The firstpenclinal division (2) is visible at this turity,these PPL cells will formthe horizontalbars on stage as well (PI in fig. 2D). The firsttransverse di- the inside of each of the outer teethand on the outer vision plane (3) is only visible in longitudinalsection surfaceof each of the innerteeth. The finalheight of and separates the segmentinto basiscopic and acros- each peristometooth is about 10-12 cells, which are copic halves (I in fig. 2E). The most frequently in turnderived fromtwo to three differentsegments viewed plane is 900 to the orientationof the individual (fig. 3F, F'). Additional transversedivisions are also segments (figs. 2E; 3A, C, D). Segment boundaries presentin the epidermaland adjacent layers at the lev- cannot be seen in sagittalsections (fig. 2F). el of the annulus (fig. 3F). These take place late in Transversecell divisions do not occur synchronous- developmentas a series of rapid divisionswithout sub- ly throughoutindividual segments.Division planes 5; sequent increase in cell size. At maturitythere is a 7a, 7b; and 13a, 13b are firstseen in the basiscopic band of specialized annular cells along which dehis- half of the segment and progressquickly toward the cence of the operculum will take place. This dehis- more acroscopic regions (figs. 1; 3A, C). Maturation cence cuts off the threeoutermost layers of cells and is thereforeacropetal in each segment.These divisions exposes the matureperistome teeth. contributeto growthin heightof the capsule, or peri- Early in development,the apex and theyounger seg- stome teeth,depending on the positionof the segment mentshave a spiral appearance on theirsurface layers undergoingdivision (segment3, fig. 3C). At the con- (fig. 3B). All sporophytesexamined have spirals that clusion of division plane 13 (13a completein fig.3E), rotatein a counterclockwisespiral when viewed from the segmentis eight cells high. above. Spiraled cell files in the surface(fig. 3B), sub- The final transversecell division plane, 16, occurs surface,and peristomiallayers persistthroughout de-

Fig.2 Surface views of the sporophyteapex of Funaria. A, Apex of young sporophyteviewed fromabove. The youngestsegment (SI) is on the leftand has undergonea single anticlinaldivision. AC = apical cell. B, Side view of A, showing relativepositions of segments1 and 2. Verticalzigzag line separates the two files of segments.AC = apical cell, SI = youngestsegment, S2 = second youngestsegment. C, Optical cross section of apex with the youngestsegment in focus. The firstanticlinal division (Al) has already taken place. The apical cell cannotbe seen because it is not in the plane of optical section.D, Optical cross section throughthe second segment.The firstpericlinal division (PI) separatesthe endothecium(EN) fromthe amphithecium(AM). E, E', Surface view of early sporophyteapex. Cell files at this stage are not spiral. Segments in the proximalportion of the apex have completedthe firsttransverse division. I = firsttransverse division of the segment,2 = second anticlinaldivision. Segmentboundaries and positionsshown in E'. F, F', As in E, viewed in sagittalplane. The firstaligned anticlinaldivisions formthe longitudinalline in the centerof the sporophyte.Segment boundaries shown in F'. Scale bar (in A) = 50 ,urm.In this figureSI is the youngestsegment illustrated, not the firstformed.

Fig.3 Cell divisions withinsegments of Funaria. A, A', Median section of a mid-developmentapex. Most of the segmentshave few internaldivisions. Segment boundaries are shown in A'. S5-SJ4 = segments5-14. B, Surface view of A, showing spiral cell files. This spiral arrangementis not found in any othergenus in the Funariales. C, C', A sporophyteat a later stage than B. Older segmentshave a four-layeredamphithecium. As developmentproceeds, the segmentboundaries (shown in C') become more horizontal.AC = apical cell, PPL = primaryperistomial layer, EN = endothecium,AC= apical cell. D, As in C, but with a five-layeredamphithecium. E, Capsule approachingmaturity. All six amphitheciallayers have been formed.Segmentation is still visible. The apical cell has long since ceased to divide regularly,and now has several internaldivisions. Deposition of peristome-thickeningmaterial has not yettaken place. Arrowindicates level of the annulus. F, F', Nearly maturecapsule of Funaria. Segmentationis visible and deposition of peristome-thickeningmaterial is beginning.The cells of the annulus are beginningto swell. Two whole segments(8-10) contributeto the peristomeon the right,while one whole segmentand partsof two others(7?/2- 1? /2) are involved on the left (see F'). S6-SJ4 = segments6-14. A, B, Scale bar (in A) = 50 ,urm.C-E, Scale bar (in C) = 50 ,urm.F, Scale bar = 100 ,urm.

Fig.4 Cell division patternswithin the segmentsof .A, A', Frontalsection of a young spear-stagesporophyte showing the apical cell (AC) and the firstsix segments.The amphitheciumis two-layered,and the more proximalsegments have just completeddivision plane 7a. Segment boundaries shown in A'. 7-14 = segments7-14. T, = firsttransverse division, T2 = second transversedivision. B, B', Sagittal section of a slightlyolder spear-stagesporophyte. The verticalline (Al) is the firstanticlinal division in each segment.Segments are shown at the conclusion of division plane 8. Segmentboundaries shown in B'. AM = amphithecium,EN = endothecium,Tr = transverse division. C, Older spear with a four-layeredamphithecium. Each IPL cell is one-halfthe heightof each PPL cell, and the apical cell has divided recently.Tr2 = second transversedivision. D, D', A completelydeveloped spear,but priorto swelling of the capsule. All fivelayers of amphitheciumare present.The apical cell has ceased to divide for some time. Althoughno depositionof tooth-formingmaterial will take place, the walls of the PPL appear thickerthan otheramphithecial walls. E, E', Surface view of a sporophyteslightly younger than D. The centralline separatingthe segmentsis not spiral as in Funaria. Segment boundariesshown in E'. T3 = thirdtransverse division. Scale bars = 50 ,urm. 240 INTERNATIONAL JOURNAL OF PLANT SCIENCES

i a i | ^ | ~~~AC_~

_s ~~~~~~~~~~~PPL

So SrF A! SCHWARTZ-FUNARIA APICAL SEGMENTATION 241

Ta _ Tr

Al _ . ~~AM

E N

_ T r Tr22 I

s | ~~~~PPL s

iT~~3 |4 A 242 INTERNATIONAL JOURNAL OF PLANT SCIENCES

- AC- AC~A

i | AE |~~~~~~~~~~~~~~I EN

x S~~~~~~~A

AS T-A

A SCHWARTZ-FUNARIA APICAL SEGMENTATION 243 velopmentand resultin the wall thickeningsthat form segments.Within a segment,most periclinal and an- the peristometeeth spiralingtoward the apex at ma- ticlinal divisions occur first,and then the transverse turity.Lower regionsof the capsule, at the level of the divisions take place. At the end of developmentthe spore sac and below, are not spirallyarranged. apical cell divides twice periclinallyand is incorpo- As the sporophyteapproaches its matureheight, the rated into the operculum. These final derivativesas- apical cell ceases to divide and divisionsbegin to take sume the shape of the othercells in these layers,and place in the youngersegments. Formation of the sec- the threeopercular layers become continuous. ond and thirdlayers of amphithecium,which previ- ously occurred in segments 6-8, now takes place in DEVELOPMENT OF PHYSCOMITRIUM segments3 and 4. In this way developmentappears to Orientationof divisions withina particularsegment "catch up" withthe apical cell. Cell divisionproceeds are identical to those observed in Funaria. However, rapidlyin the region thatwill differentiateinto the urn the timingand sequence of certaincell divisions are and sporogenous layers. As this growthoccurs, mat- slightlydifferent. The firstseries of transversedivi- urationof the more basal, peristome-formingsegments sions, which divide the segment into basiscopic and takes place. Late in maturityall of the segmentsnear acroscopic halves (division plane 3, fig. 4A, B), pre- the apex have several internaldivisions (fig. 3D-F). cedes the second series of radial divisions (division The epiphragmis the most distal portionof the col- plane 6). This transversedivision plane includes the umella and is derived fromthe innerportions of seg- synchronousdivision of six separate cells, and at its ments 11 and 12. The divisions that form the epi- completion all of the resultingcell walls are aligned phragm resemble those that occur early in the precisely.These two divisions (4 and 3) occur in the developmentof the amphithecium(fig. 2D). opposite orderin Funaria. Althoughall segmentsundergo a similarpattern of The next series of transversedivisions begin in the divisions,only the morebasal segmentsthat contribute outer layer of the amphitheciumand proceed to the to the peristometeeth complete the entire sequence. IPL, dividing each of the cells of the segment into Younger segments,i.e., 11-14, consist of fewer cells basiscopic and acroscopic halves (13a-d) (fig. 4D, at maturity(fig. 3E, F). For example, segment12 has and T3 in fig.4E). The layer thatforms the PPL does six layers of amphitheciumin its basiscopic half but not divide transversely.The divisionbegins in thebas- is only fourcells in height.The peristometeeth end at iscopic portionsof the segmentand proceeds acrope- the base of segment 11 on one side of the sporophyte tally. Unlike other divisions, this particularone does and at the midlevel of segment 10 on the other side not occur synchronouslythroughout the segment. (fig. 3E, F). In the youngest three segments,fewer Transversedivisions of the IPL occur much later and amphitheciallayers are formed.Segment 11 consists are complete by the completionof division plane 12. of six amphitheciallayers at its basiscopic surfacebut In total, division plane 13 involves the separate divi- has only four at its acroscopic end. Segment 14 has sions of 48 cells and is not visible in cross section. only threeamphithecial layers throughoutits height. The latercell divisionplanes returnto the generalpat- These segmentsdivide as expected but cease to di- ternof alternatinganticlinal, periclinal, and transverse vide at division plane 12 for the acroscopic end of divisions,as in Funaria. segment 11 and division plane 8 for segment 12. All The final division of the segmentis one in which of thesemost apical segmentsdivide manyfewer times the cells of the surface layer divide transversely,re- in the transverseplane. Segment 13 has five layers of sultingin the segmentbecoming 16 cells in height(fig. amphitheciumbut only one transversedivision. Seg- 1, division plane 18). As this division plane "moves ment 14 has three amphitheciallayers but only one acropetally," cells of the OPL also divide in a trans- transversedivision. The innermostcells of theseyoun- verse plane. These cell divisionplanes are aligned pre- gest segments,homologous to cells that will become cisely at the completionof division. endothecium,do not divide. In segment 12 the en- Late in developmentthe individualcell filesbecome dothecial initial is divided only by a single transverse distortedby the swelling surface layer cells. At this wall. stage of development,positions of the segmentscan The timingof thesetransverse divisions in theyoun- only be determinedby countingsurface cells and in- ger segmentsis also differentfrom those in the older ferringsegment boundaries. Although the development

Fig.5 Cell division patternswithin the segmentsof Aphanorrhegma.A, A very young spear-stagesporophyte of Aphanorrhegma.The apical cell is large and actively dividing. Segments are presentin portionsof the sporophytethat will formthe capsule. AC = apical cell. B, B', A sporophyteolder thanA, with a three-layeredamphithecium (AM). AC = apical cell. Segmentboundaries shown in B'. S4-SII = segments4-11. EN = endothecium.C, An older capsule with a four-layeredamphithecium. The endothecium(EN) has begun to divide. D, D', The apex of a sporophyteat the conclusion of division. Segment boundaries shown in D'. E, E', Early capsule swelling. The cells in the more proximalportions have begun to enlarge. The line indicates the level of the annulus,which is slightlybelow the top of the spore sac. AS = the position where the air space will form.F, Longitudinalsection of a maturecapsule. The segmentboundaries and apical cell are no longer distinguishable.The air space has enlarged,and the annulushas differentiatedwhere the line indicatingthe spores (S) crosses the capsule. A two-layerIPL formsthe spore sac. Scale bars = 50 p1m. 244 INTERNATIONAL JOURNAL OF PLANT SCIENCES of Physcomitriumis virtuallyidentical to that of Fu- cating that the apical cell divides only slightlyless naria, no spiralingof cells takes place. Both early and frequentlythan in Funaria. In early embryos, most late spear-stage sporophytes, examined in surface segmentsare undivided or have a single vertical an- view, have cells aligned in verticalfiles, not in spiral ticlinalwall (fig. 6A, B). Growthof the capsule results rows (fig. 4B, E). exclusively frominternal divisions withinthese nine Between the annulus and apical cell there are six segments. At a slightlyolder stage the more apical segments,and of these segments, 12, 13, and 14 do segmentsare undividedwhile the basal segmentshave not completethe entiresequence of cell divisions (fig. two to threeinternal divisions (fig. 6B). 4D). Segment 14 expands as a single amphithecialcell The division sequences within each segment of and a single endothecialcell. Divisions in segment13 Ephemerum are virtuallyidentical to early division cease afterthe amphitheciallayer has divided trans- planes withinthe segmentsof Funaria, except forthe versely. Segment 12 has two amphitheciallayers and timingof the transversedivisions. The firsttransverse is fourcells in height(fig. 4D). division (3) of the segment occurs afterthe second radial division and occurs only in the endothecium.It DEVELOPMENT OF APHANORRHEGMA is later,after the amphitheciumhas been divided into The young embryos of Aphanorrhegmahave eight two layers by the second periclinal division (5), that segmentsabove the small-celled seta and foot regions this transversedivision plane (3) is completed in the (fig. 5A). A total of 11 segmentsare cut offfrom the amphithecium.There is no differencein theorientation apical cell duringthe developmentof the sporophyte. of these planes of division fromthose in Funaria; the The more apical segmentsare orientatedat increasing- only differenceis in theirrelative timing. A transverse ly more verticalangles. The cell divisionpattern with- division does not precede the periclinal division that in an individualsegment is identicalto thatof Funaria formsthe thirdring of amphithecium,unlike in Fu- but terminatesfar sooner.At maturitya segmentat the naria. Afterthe radial division of this thirdamphithe- level of the annulus has only fouramphithecial layers. cial layer,the segmentis only two cells high. Therefore,divisions within a maturesegment cease af- The final transversedivision (7a, 7b) divides both ter the radial division of the fourthamphithecial layer the basiscopic and acroscopic halves of the segment (12 in Funaria). All segmentsabove the annulushave into basiscopic and acroscopic quarters,but it occurs threeamphithecial layers or fewer (fig. 5B, D, E). At only in foursegments (segments 7-10). The more api- the level of the annulus, the epidermal cells divide cal segments do not increase in height beyond two transversely;therefore, at maturitysegments that con- cells (fig. 6C, D). The apical cell remains as a point tributeto this region of the capsule are six to eight at the top of the capsule (fig. 6F). Especially notice- cells high (fig. 5E, F). able duringcapsule expansion is the formationof the The youngersegments of the capsule do not com- air space surroundingthe spore sac, although this is plete the entiresequence of cell divisions observed in presentin all taxa examined. This firstappears as a the more basal segments(fig. 5E, F). Segment 11, the dark line between the IPL and PPL (fig. 6D). In older last formed,is the least developed, and consistsof two capsules a large air space is seen withthe cells of the amphithecialcells and two endothecialcells. Segment PPL, which assume a spherical shape, projectinginto 10 is more developed; it consists of two layers of am- the open space. Subsequent to air space formation,the phitheciumand is two cells high. Segment 9 has two IPL divides periclinallyso thata separatetwo-layered layers of amphitheciumand is three cells high. Seg- spore sac will be formed,but only in the more basal ment 8 nearly completes the entiresequence of divi- segments. sions. All of these more apical segments(8-11) have The youngersegments in the capsule do not com- thin-walledendothecial tissue thatbreaks down at the plete the entiresequence of divisions as do the older time of capsule maturity. segments.Segment 9 is only one cell high but is com- posed of three amphitheciallayers. Segment 11 per- DEVELOPMENT OF EPHEMERUM sists as a single cell throughoutdevelopment (fig. 6A- The young embryosof Ephemerumare oval in out- D). Furthermore,cell division sequences are frequently line and are considerablysmaller than are the embryos irregularlate in maturationin more apical segments. of Funaria at a comparable stage in which an equiv- Cell divisions are not as predictable,and examples are alent numberof apical cell segmentsare formed.The found where threepericlinal divisions take place be- entire sporophyteis composed of 11 segments,indi- fore any transversedivisions occur. At maturity,be-

Fig.6 Cell division patternswithin the segmentsof Ephemerum.A, A', A very young embryowith seven segments.Segment boundaries and internaldivisions are shown in A'. The apical cell will divide two more times before stopping.AC = apical cell, F = foot cell. B, B', A slightlyolder embryo.A two-layeramphithecium (AM) is alreadypresent in the more distal segments.Segment boundaries are illustrated in B'. EN = endothecium.C, An older sporophyteviewed at 900 to A, B, and D. A two-layeramphithecium (AM) is present.D, A mature capsule. The segmentboundaries and the apical cell (AC) are still visible. There is a three-layeramphithecium, and the IPL is divided in two where it formsthe spore sac. The air space (AS) has already formed.An annulus is not present.A, B, Scale bar (in A) = 50 P'm. C, D, Scale bar = 50 p1m. SCHWARTZ-FUNARIA APICAL SEGMENTATION 245

AC ______......

S9SS1 S7 S5 A 246 INTERNATIONAL JOURNAL OF PLANT SCIENCES cause of cell expansion and formationof the air space, divisions of the IPL, form walls that are exactly no segmentboundaries can be observed. aligned with existingcell walls in the OPL and PPL. This is, however,only knownthus far in theFunariales Discussion and Polytrichales,since in nearly all othertaxa these Not since the last centuryhave questions of seg- walls are unaligned. Control of alignmentof the final mentationand its relationshipto peristometooth for- division of the IPL with a similarwall in the OPL has mationbeen fullyconsidered. The "fundamentalquad- not been explained. It mightbe suggestedthat this is rants" describedinitially by Hofmeister(1862), Kuhn just the resultof the cells dividing into equal deriva- (1870), and Goebel (1905) have been reportedfor all tives withina series of concentriccircles. The resulting so far examined and, in fact, in many other alignmentwould then be the result of geometry,not generaof plants.This stage resultsfrom a cross section of developmentalcontrol. This would not explain the throughthe two youngestsegments, each divided once situationin Bryum and the Haplolepidae where late anticlinally(fig. 2C). It is very difficult,however, to divisions of the IPL are asymmetrical(Shaw et al. determinewhich cells are in the same segment.The 1989). Similarly,in Bartramia, the firstlate division four-cellstage is not seen frequentlybecause it is nec- of the IPL is nearly symmetrical,while the next late essary to cut a plane of section throughthe basiscopic divisions are more asymmetrical(Saito and Shimoze half of the youngestsegment and the acroscopic half 1954). of the next youngestsegment. This region of overlap is fairly small. Furthermore,these segments are not DIVISIONS WITHIN A SEGMENT orientedtransversely but rathersteeply, parallel to the The sequence of cell divisions withineach individ- cuttingfaces of the apical cell. Therefore,cross sec- ual segmentfollows a common pattern.All segments tions at this level are frequentlyoblique with respect in Funaria appear to follow exactlythe same sequence to the segments. of cell divisions that is illustratedin figure 1. Early Cell divisions at the apex follow an extremelyreg- segmentscut offfrom the apical cell, which will form ular and precise patternas segmentsare cleaved off. the seta, also have initial division planes similar to Similar patternsof apical cell segmentationhave been those in segmentsthat will contributeto the peristome. found in other small axes formedby divisions of a In segmentsforming the seta, all amphithecialcell lay- single apical cell, e.g., Azolla (Gunning et al. 1978). ers have equal numbersof cells. This arrangementis Fern apices, however,have apical cells withthree cut- presentin sectionsbasal to the spore sac, where a dif- tingfaces and consequentlyhave threevertical files of ferentconclusion to the developmentalprogram is ob- segments. served. Cells of the sporophytecan be traced to divisions Segments that will contributeto the apical half of of a single apical cell, which is primarilyactive early the capsule are cut off early in developmentand re- in development,cutting off a total of about 14 seg- main with few internalcell divisions until seta elon- ments,the 10 most distal being responsiblefor capsule gation is almost complete. At this point segmentsdi- formation.Although the capsules of Ephemerumand vide according to the sequence already described. Funaria differgreatly in size, only three fewer seg- Divisions begin in thebasal segmentsfirst and proceed ments are formedin Ephemerumcompared with Fu- acropetallyto the more apical segments.The youngest naria. The threesegments immediately above the foot segments are frequentlyundivided when segment 7 are responsiblefor the growthof the seta in Funaria. has formeda three-layeredamphithecium. Rather than Differentiationof the:intercalary meristem region is examining young plants, early workers serially sec- not observed in Ephemerumor Aphanorrhegmaand tioned nearly mature capsules. Therefore, sections would indicate that the lack of the intercalarymeri- throughthe apical portionsof a rathermature devel- stem,and not a greatlyreduced numberof apical cell oping sporophytewere frequentlyreported as early segments,is responsiblefor the morphologyof these stages. The sporophyteis a determinatestructure, and sporophytes.Furthermore, we conclude thatlack of an it was previouslyunknown whethersegments devel- intercalarymeristem is a derived condition in these oped identically.Because it now appears thatearly de- two taxa, since presence of one is shared with distant velopment of the segments is identical, most of the outgroupssuch as the Polytrichales. previousobservations seem to be justified.Because the Early in development,all cells withina segmentdi- sporophyteis determinateand maturesacropetally, de- vide in a given plane at nearly the same time. While velopmentappears to catch up withthe apical cell once control of this process is not known at this time, it it has ceased to divide. As maturityapproaches, the should be noted that as the segmentbecomes larger, apical cell itselfdivides periclinallyand in this way is the timingof divisions becomes less regular.For ex- incorporatedinto the opercularlayers. ample, formationof the second ring of the amphithe- In all taxa the most distal segmentsdo not complete cium occurs synchronouslyaround the perimeter of the the entiresequence of divisions thattake place in the sporophyte,while formationof the sixth ring of am- segments that form the lower portions of the peri- phitheciummight occur duringthe anticlinaldivision stome. For example, later anticlinal divisions of the of the fifthlayer on the opposite side of the capsule. IPL and the periclinal division of the IPL into two Furthermore,late divisions, such as the two later layers do not take place in the more apical segments. SCHWARTZ-FUNARIA APICAL SEGMENTATION 247

For this reason these divisions were oftenmissed in numberof cells on the surfacedown fromthe remains earlierworks. Blomquist and Robertson(1941) clearly of the apical cell. In Funaria, the verticalfiles of cells examined a wide range of stages and showed for the smoothlyspiral across verticalsegment boundaries to- firsttime thatthe IPL does divide anticlinallylater in ward the apical cell. In all other taxa, the files are development.This would not have been seen if they orientedvertically. Because the peristometeeth cross had examined the apical portionsof more maturecap- segment boundaries and are thereforecomposed of sules looking for earlier stages. cells derived from differentsegments, it is apparent Cell division planes are coordinatedbetween hori- thatcell lineages are of limitedimportance in guiding zontally adjacent segments.Throughout the literature differentiation.Instead, both position within a segment referencesare frequentlymade to stagesas seen in cross and withinthe sporophyteas a whole determinesthe section (Blomquist and Robertson1941). For example, fate of any given cell. a stage wherethree amphithecial layers are presentand the outermostlayer is dividinganticlinally might be il- SPIRALING OF CELLS IN FUNARIA lustrated.However, in all such cross sectionstwo seg- In all taxa examined, the vertical files are parallel ments are shown, each formingone-half of the seta. to the axis of the sporophyte,but in Funaria the files This was noted by Campbell (1913) but was not ex- are steeply spiraled. The process of spiralingbegins amined in any detail. It is not possible to determine long before capsule expansion takes place and results which two quadrantsform a single segment.Each of in spiraled peristome teeth at maturity.When only these segmentsis offsetone-half a segmentin height three amphitheciallayers are present,a spiral pattern with respect to the next oldest or youngestsegment. is visible in surfaceviews of cleared sporophytes(fig. Therefore,the acroscopic half of the older segmentis 3B). The spiralpattern may be the resultof differential directlyopposite the basiscopic half of the next youn- expansion of cells, since there is no increase in cell gest segment.These two portionsof segmentsare al- number as the spiral forms. In median longitudinal ways coordinatedin theirtiming of cell divisionssuch section a spiral is not evident,with the apical cell vis- that a given cell division plane appears to occur syn- ible in section with two verticalfiles of segmentsori- chronouslyaround the entiresporophyte. In longitudi- ented directlyopposite each otherbelow (fig. 3A). All nal section a new ringof amphitheciumis always seen of the segmentsappear to be in median section. The to be formingon both sides of the seta at a given level centralline thatseparates the two files of segmentsis (fig.3A, C, D). For example,as one segmentcompletes not spiraledin median sectionbut shows the usual "z" the formationof the fourthring of the amphitheciumin appearance. However, in surface view this same cen- its acroscopic half (11), the nextyoungest segment has tral line is spiraled (fig. 3B). Therefore,the segments completedthis division in its basiscopic half. This ap- are rotatedaround the axis, since the cell files cannot pears to be positional controlof acropetalmaturation, cross the segmentboundaries. One possible explana- perhapsunder hormonal control by the apex. tion for this arrangementof segmentsis thatafter the Peristome teeth extend verticallythrough segment apical cell cuts offall of the segments,differential cell boundaries that are perpendicularto the axis of the expansion takes place and the segmentsare forcedinto sporophyte.Peristome teeth of Funaria traversetwo the spiraled orientation.Since thereare far more cells to three segmentsfrom the annulus to the top of the expandingat the surfaceof the sporophytethan in the columella. On one side of the sporophytethe peri- midplane, the rotationis greatestnearer the surface, stometeeth begin at the base of segment8 and traverse and near the centerof the axis almostno rotationtakes two segments.On the opposite side of the sporophyte place. In othergroups of mosses spirallyarranged peri- the peristomebegins at the midline of segment7. Be- stome teethare quite common,but thesehave notbeen cause this tooth begins at the midlevel of a segment examined here. In all taxa otherthan Funaria exam- ratherthan at a segmentboundary, it also ends at the ined in thisstudy, the segmentsremain in thepositions midlevel of a segment,which is two segmentshigher in which theywere cut offfrom the apical cell. on the same side of the sporophyte.The uppersegment Spiraling of segmentsaround an axis has been re- of each tooth was cut off by the apical cell four di- ported in ferns (Davallia, Dipteris) (Bierhorst 1977), visions later than the segmentforming the lower por- where spiraling apparentlytakes place graduallybe- tion of the tooth. This tooth crosses two segment cause the apical cell cuts off segments at an angle boundaries and portionsof three segments.A single slightlyoblique to the existingcutting face. Aftersev- toothnever formsfrom adjacent cell filesthat are part eral rounds of division the apical cell will appear to of two differentsegments. Rather, four teeth are have rotated.Similar slightlyoblique divisions of the formedwithin each of the originalfour quadrants. apical cell may also be responsiblefor the spiralingof At the points where peristometeeth cross segment cell files in Funaria. Because each apical cell division boundaries,there are neitherlateral displacementsnor is only slightlyoblique, in median longitudinalsection irregulardivisions. The vertical files, or layers, are the younger segments appear to be at the same ori- alignedperfectly between segments. No segmentbound- entationas the adjacent older segmentin the same ver- aries are visible when thickeningbegins. However,be- tical file. However, in the surfacelayers of the sporo- cause the cells withinthe segmentsare no longer di- phyte, where there are a greaternumber of cells, a viding, boundaries can be identifiedby countingthe greaterdegree of rotationis observed. 248 INTERNATIONAL JOURNAL OF PLANT SCIENCES

SYMMETRY IN DEVELOPMENT said to be radially symmetrical.However, the apical cell does not cut off two derivativessimultaneously, describedthe capsule of Mittenia Stone (1961, 1988) one fromeach cuttingface. There are many radially as being unique among the mosses in being bilaterally symmetricalplants derived fromapices with a single symmetrical,while all othermosses are radiallysym- apical cell. However,in these cases, the apical cell has metrical.In fact,the capsules of all mosses are bilaterally threecutting faces (Schuster 1966). symmetricalas a resultof theirgrowth pattern. Stone's Because of the alternatearrangement of the seg- statementis based primarilyon theirregular cell division ments as seen in longitudinalsection, the sporophyte planes of Mitteniathat first divide each of thequadrants mustbe bilaterallysymmetrical. Only a plane dividing into endotheciumand amphithecium.The firstdivisions the sporophytelengthwise perpendicular to the seg- would give the appearance in each quadrantof Mittenia mentswould resultin two mirror-imagehalves. There- of a bilaterallysymmetrical structure. Similar cross sec- fore,Stone (1961) is correctin thatthe sporophyteof tions of Funaria or any othergenus examined in this Mittenia is bilaterallysymmetrical, however, not only studywould indicatea radiallysymmetrical structure be- for the reasons suggested. cause the division(2) is pericinal and identicalin each of the fourquadrants. Although the maturecapsule of Acknowledgments Funaria is radially symmetrical,the symmetryof the sporophytecan only be understoodcompletely from ob- I wish to thankL. E. Anderson,B. D. Mishler,M. servationsof longitudinalsections. D. Turner,and R. A. White forhelpful suggestions and In a longitudinalsection the apical cell can be seen discussion duringthe course of thisresearch and B. E. to have two cuttingfaces; therefore,there are two ver- S. Gunning for editorial assistance with the manu- tical files of derivatives,offset with respect to each script. I also wish to acknowledge support through other.If the derivatives were directlyopposite each NSF grantBSR 8506992 to L. E. Anderson,J. Shaw, otherin the seta and capsule, a plane parallel to each and B. D. Mishler.This work was submittedin partial of the quadrantwalls would divide the sporophyteinto fulfillmentof the requirementsfor the Ph.D. degree in two identical halves. The sporophytecould thus be the Departmentof Botany,Duke University. Literaturecited

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