Osterr. Bot. Z. 122, 99--114 (1973) �9 by Springer-Verlag 1973

Dehiscence of the Moss Capsule

I I I . A n u l u s F u n c t i o n a n d t h e L i d S t a b i l i t y : a S t u d y w i t h t h e L i g h t - a n d S c a n n i n g E l e c t r o n M i c r o s c o p e

By

Kurt Maier, Montreal

With 19 Figures

(Received March 2, 1973)

There is a local change in the character of the cells where the lid meets the urn. Here, the epidermis--al though being integral par t of the capsule exoskeleton--must also function as the mechanism of dehiscence (MAIE~, 1967). The present paper illustrates how the need to fulfill these contradictory functions results in strains.

General Features of the Material

Mature capsules dehisce almost instantly in the presence of liquid water, once there is an opening between lid and anulus. The action is so fast in mature capsules, tha t direct observation of the dehiscence phases is nearly impossible. However, the tissues loosen gradually during maturat ion and the critiOal point--where dehiscence occurs ins t an t ly - -may be reached only one or two days prior to dehiscence. Therefore until a short t ime before tha t critical point, it may be possible to observe capsules undergoing dehiscence bit by bit; sequence photo- graphs would show a kind of "slow-motion" dehiscence.

Such premature dehiscence can be triggered by allowing water to enter the zone of weakness through a short slit tha t is being made between the lid and the anulus. Of course, care must be used to record only the typical and natural features of the dehiscence process--but little experience is needed to decide positively in each case. The only real problem is to come upon the capsules at the right moment, that is, when the line of dehiscence is loose enough to allow natural dehis- cence to occur, but not so loose tha t the lid springs back, making the detachment hardly observable without high-speed photography equip-

7*

100 K. MAIEB :

ment. The solution--easy, but somewhat time-consuming--is to watch the ripening capsules over a period of 40-60 hours starting 4-2 days before the dehiscence would normally occur.

Preparat ion Methods

The sporangia of Mnium cuspidatum L. dehisce in the Montreal area on May 20 • one to three days depending on weather conditions. Fruiting material was gathered May 15 and kept in the greenhouse

until maturity. Twice a day, the material was tested as follows: Several

sporangia were cut off and fastened to the stage plate of a Wild M 5

microscope. The zone of weakness was gently tapped with microforceps or the tip of a needle. The degree of maturation was considered optimal when a very light tap with one of the said instruments was enough to rupture the zone of weakness between lid and anulus--this is the line of dehiscence--but not between anulus and urn. When most sporangia had reached that stage of maturity, a larger quantity of capsules were subjected to one of the procedures below.

S c a n n i n g e l e c t r o n m i c r o s c o p y . A short slit was made along the lid as is described above. The capsules, with their basal ends cut off, were then mounted with water, upright, on a small circular cover slip, care being exercised to keep the ruptured area dry. Water was then applied to the slit using a fine artist's brush, and only enough water was used to keep the dehis cence process under control.

The capsule tips were quickly pushed into cold Freon 11 (coolant: liquid nitrogen) when the desired dehiscence stage was reached. After 20 seconds, they were transferred to the cold stage of a Speedivac- Pearse tissue dryer. They remained under vaccum for approximately 40 hours, the first 24 hours at - - 80 ~ and the rest at - - 20 ~

The dry specimens were mounted singly on standard stubs, and the surfaces cleaned of adhering spores. They were then coated ~aith a thin layer of carbon, less than 50 •ngstr6m units thick and a layer of gold,

about 300 A. L i g h t m i c r o s c o p y. Two views of the capsules were made : a top view

and a profile view. Top view: The apices of fresh sporangia were cut off and

mounted upright on a slide with lanolin as the adhesive. The zone of

Fig. I. Diagrammatic representations of the capsule apex showing the distortion that it undergoes during dehiscence. Stage O was recorded from the apparently fully turgescent capsule while lying "dry" between slide and cover slip. All other stages show the capsule after water had been added. The drawings are tracings from photographic negatives. Each stage is shown twice: in the first pair drawn in heavy lines, stippled in

the following

Dehiscence of the Moss Capsule III 101

1 + 2 2 + 3 f

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3 + 4 4 + 5 ~ 5 + 6

+ 7 7 + 8 6 ~ 8 + 9

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Fig. I

102 K. MAIEJa :

weakness was then inf i l t ra ted with wate r as descr ibed above and the dehis- cence stages resorded on K o d a k Tr i -X P a n th rough a Wi ld M 5-dissect ing microscope, f i t t ed wi th a pho to - tube and au toma t i c exposure uni t . The l ight , inc ident upon the specimen, was p rov ided in sa t i s fac to ry in tens i ty b y two smal l -area l ight sources and add i t iona l ref lect ing screens convenien t ly a r ranged a round the specimen.

Profi le view: The capsule was s t ra ined the usual w a y to rup tu re the line of dehiscence, b u t was otherwise left in tac t so t h a t the wa te r in take had to occur th rough this opening. The slit length never exceeded 1/10 of the t o t a l circumference of the capsule. The capsules were then glued to slides in recl ined posit ions, a cover slip p u t on and secured wi th smal l b i ts of lanol in to keep i t f rom rocking, and to minimize pres- sure on the dehiscing capsule l a te r when wa te r was added.

The capsule was pho tog raphed th rough a Wi ld M 21-microscope using t r a n s m i t t e d l ight suppl ied b y the s t a n d a r d bui l t - in lamp. The camera was focussed on the f lank wi th the slit. T ransmi t t ed l ight gives an ex t r eme ly accura te profile view of the opaque capsule walls, bu t does no t disclose detai ls of the in te rna l organisat ion.

Capsule dimensions. Leng th : 2.5-3 m m ; d iamete r (zone of weak- ness): 0.8 ram; height of the anulus : 0.1 mm.

Observations

L

Fig. 1 shows a series of profiles of the apex of the capsule. I t s in i t ia l f o r m - - i l l u s t r a t e d as s tage 0 in Fig. 1 - - w a s redorded f rom a sporang ium which had been moun ted " d r y " between slide and cover slip, according to the m e t h o d descr ibed above. W a t e r was then added b y the edge of the cover slip unt i l br imming, while the capsule was careful ly wa tched under the microscope. The water was seen rushing into the capsule inter ionr , inducing swelling wherever i t spread a round the capsule wall.

Fig. 2. A dehiscing capsule. The photograph was taken from a freeze- dried specimen. - . . . . marks the place whore the sequential stages to Fig. 6 would have been taken if tMs were the original capsule. The camera would have been positioned to the right of this mark, facing the advancing front of the slit. This photograph also provides several surface views to the dehiscence stages of Fig. 6. These, marked by arrows, are from left to r ight : /, d, c, b, a . ~ F i g . 3. The "bulge" which forms on top of and by the action of the uncoiling anulus is seen between the two asterisks. Com- pensatory retraction occurs between the two anulus eoils . --Fig. 4. View of the unopened left-hand side of the capsule shown in Fig. 2. Note the bulging lid margin; no slit can be seen yet. This figure may be compared

with the left-hand side of Fig. 5 (see also Figs. 6a-e)

Dehiscence of the Moss Capsule I I I 103

Figs. 2-4

104 K . MAteR :

Stage 1 is the silhouette of the capsule 30 seconds after being im- mersed in water. Its changing shape gives evidence of water below the lid tip and at the extreme left-hand side. Stage 2: Water has spread to the right-hand side of the lid; swelling follows in due course. The following stage, distinguished as no 3, signals the filling to the brim of the space between lid and peristome, or perhaps the entire lid cavity. Expansion of the capsule has now reached the climax.

Only a little later, and the zone of weakness becomes the centre of activity (stage 4); expansion appears to have nearly ceased in the re- mMnder of the capsule tip. The sli t--however--is still growing in both width and length. I t may be noted in this connection that normally the lid as a whole is lifted up by about the same amount that the slit widens vertically. The present case is somewhat different: compare the summit regions of the two pairs of superimposed outline drawings-- stages 1 and 2 and in particular stages 2 and 3. The urn mouth and the lid portion of the zone of weakness are both seen to protrude signifi- cantly.

Stage 5 of Fig. 1: The anulus had previously escaped the grip of the lid; comparatively little change is noticed elsewhere, even the slight corrugation of the lid can still be seen on the left-hand side. Lid and anulus are now separated by a narrow slit (see stages 5-8): the centres of stress must therefore lie outside the focal plane of the camera.

The following stage 6 shows a strongly reclining anulus with the

outer periclinM walls arched. The lid is still in its former position. The arching is undoubtedly a sign of stress which developed alongside several other such signs elsewhere: the convex shape of the left-hand side, and the three or four shallow depressions that roughen the right- hand lid side.

Stages 7 and 8. The lid has assumed the highest position ever, and now the long expected happens. The lid margin snaps back to what

is believed to be the initial position. Stage 9. Still the slit widens and gradually another distortion pattern

begins to take shape. The loose side of the lid is again involved although its margin is separated from the appertaining part of the anulus im- mediately beneath. For explanation see chapter I I I and Fig. 3.

II.

In a second series of experiments the camera was focussed on the still unruptured area a little ahead of the slit in order that the initial

dehiscence stages could be seen. The preparatory and recording methods were the same as before, except that the slit was made at the far side

Dehiscence of the Moss Capsule III 105

of the capsule. The slit grew in both (tangential) directions when water was added, its length totalling about 2/3 of the capsule circumference when the proximal of its end finally came in sight. Fig. 2 (see also Fig. 7), which is a scanning micrograph of another capsule, may give an idea of the capsule's appearance when the series of stages of Fig. 6 were recorded.

Fig. 4 reproduces the far right-hand side of the same capsule. I t shows how the unopened flank must have looked before the taking

Fig. 5. Diagrammatic profile view of the capsule in Fig. 2. This diagramm is intended to explain the relative positions of lid, urn and anulus, while the capsule dehisees. The undetached part of the anulus functions like a hinge. This short, nearly straight piece of the anuhis can topple over without severing the connection with the lid and the urn, respectively. The result is seen in this diagramm: The l id as a whole is being dragged

toward the "hinge"

of the sequential pictures to Fig. 6. Note tha t the lid bulges out, al- though no trace of a slit can be detected yet. This phenomenon is explained in the caption to Fig. 5.

Stage a of Fig. 6 and Figs. 2, 7, and 8 illustrate how the zone of weak- ness looks when the anulus is slowly being squeezed through the epider- real bottle neck between lid rim and urn lip. The curvature of the anulus apex (see encircled area) suggests that pressure is high when this happens.

Stage b of Fig. 6 and Fig. 13: The slit has grown in length and width, releasing a corresponding portion of the anulus apex. The features

106 K. MAIER: Dehiscence of the Moss Capsule [ I I

are basical ly as t hey were in the previous stage, except t h a t the l id was pushed up a p p r o x i m a t e l y 14~o as compared wi th s tage a and the t i l t of the anulus is now 35 ~ New and r emarkab le are the following character is t ies : (1) the warp ing marg in of the l id and (2) i ts deformed r im (arrow) which is known to be less incrus ted t han the r ema inde r (see paper I I of the present series).

Stage c (lid ra ised b y 7~o; t i l t angle: 38~ Here the anulus is stil l j a m m e d between the lid and urn (Fig. 13), exer t ing i ts pressure diag-

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Fig. 6. Sequential dehiscence stages. Measuring marks are seen below" and above the zone of weakness. The t i l t angle of the anulus is the deviation

from the vertical position

onal ly on bo th of them due to the grea ter t i l t . The urn is a p p a r e n t l y s t ronger t han the lid, which d isp lays f rom the beginning of these events a eertaiI1 ins tabi l i ty . But the urn ' s s t rength changes suddenly. I t gives way when the pressure of the anulus is exer ted ful ly t o w a r d the side. The first s ignificant dent occurs jus t benea th the junc t ion wi th the

Fig. 7. Short piece of the capsule displaying a spatial sequence of dehiscence stages from the earliest a t the left-hand side to the latest at the opposite side. This photograph provides the surface views of several stages of the previous Fig. 6. The same labelling has been applied to bo th . - -F ig . 8. This photograph shows the "bulge" labelled ~ according to stage c of Fig. 6. Stage / is shown to the left. Note tha t the lid margin has snapped back there, and also tha t the lid rim cuts into the apex of one of the anulus ceils

Figs. 7~8

108 K. MAIE~: Dehiscence of the Moss Capsule I I I

anulus, and also the outer periel inal walls of the anulus are seen to suffer deformat ion at this stage. All this happened in the here descr ibed exper iments when the anulus t i l t was be tween 35 ~ and 38 ~

Stage d of Fig. 6 and Fig. 12 (@ 9 % ; 44~ The anulus is shown at a last t e m p o r a r y ha l t a t the l id r im. The new t i l t - - 4 r ~ in the presen t ea se - -once more helps to increase the pressure in the zone of weakness. Note the w a v y urn wall.

Stage e and Fig. 9 ( + 2 . 5 ~ 65~ The anulus now comple te ly emerged f rom under the l id r im is ca t apu l t ed from the previous 44 ~ to a t i l t of 65 ~ rel ieving a t the same t ime the pressure f rom the l id margin. The l a t t e r snaps back to the nea r -normal posi t ion shown in the present stage, bu t the overal l t i l t of the l id remains p rac t i ca l ly unal te red . Still the l id is rising up modera te ly .

The slit w id th declines b y - - 1 0 % in the following s tage f . The urn deformat ion becomes comple te ly reversed, bu t this new convex p a t t e r n is only shor t - l ived (compare wi th the nex t s tage g). The ou te r periel inal walls of the anulus, here also known as the " b a n d of resist- ance" , once more suffer de format ion ; note the S-shape of the profile. This s igmoid form is ce r ta in ly the resul t of the anulus ' fai lure to conform to the new recl ined anulus posi t ion b y d i la t ing the d is ta l side of the f i rm band of resistance.

Stages g-h, see also Figs. 10-14: The zone of weakness appeares now st ra imfree.

Stage i : The lid assumes ye t ano ther posi t ion re la t ive to the urn. I t seems t h a t the l id marg in becomes s l ight ly d rawn inward. One can wonder in wha t w a y this new p a t t e r n is re la ted to the ac t ion of the de taching anulus.

Figs. 9-11. Three electron micrographs show how the emerging anulus affects the form of the lid. The following pictures were taken from freeze- dried ma te r i a l .~F ig . 9. This first picture is an early stage. Note tha t there is only a single bulge from which the anulus is just breaking free (see in part icular the left-hand side) . --Fig. 10. The original bulge is divided into two daughter project ions . - -Fig , l l . This third picture may serve to il lustrate stage c of Fig. 19: The "notch" and the two flanking bulges are clearly diseernable (lower r ight-hand side) . --Fig. 12. Profile view of a capsule. The critical stage d of Fig. 6 is seen at the extreme r ight-hand side. Anulus crest and lid rim have slightly separated due to the handling of the freeze-dried material. F rom right to left: dehiscence stages d~/ . - - Fig. 13. Same capsule as in Fig. 12, slightly turned to the left. Profile view

of stage b (Fig. 6) .--Fig. 14. Capsule showing dehiscence stages a - i

Figs. 9-14

i10 K. MAIEg:

III. Photographs were taken of the anulus region of the dehiscing cap-

sules in an at tempt to determine the horizontal range of the previously established turned-up portion of the lid (Figs. 15 and 16). Detailed account of the method was given under "Preparation methods." The results confirm that the lid margin undergoes a series of rather conspicu- ous and reversible changes (Figs. 15 and 16).

The first visible changes occur only seconds after water is allowed to enter through the artificially made slit. Soon the zone of weakness begitls to swell up around this slit (Figs. 15a--e) and a well defined pro- jeetion appears (Fig. 9). This bulge, being inconspicuous at first, soon gains in length as the slit extends tangentially and vertically as well, eventually allowing a comparatively short length of the anulus to break free from the rim of the faintly nose-like projection (Fig. 15b and Fig. 9). Nine out of ten anuli endure that " t reatment" without breaking to pieces. The "nose" on top persists for the time being, but disappears as the slit fronts gradually move on (Fig. 10). The lid margin falls back (see also Fig. 15c) at the same time to reassume the former position while two daughter projections spring up to replace the single original one (Fig. 10 and also Fig. 3). These secondary bulges form close to the slit front where the annlus is about to pass through the stages a-e shown in Fig. 6.

The circumference (periphery) of the lid remains practically constant as this happens, so that any local outward shifting of any particular portion of the lid rim has by necessity to be accompanied by compen- satory retraction of others (Fig. 3). Fig. 17 shows in almost ideal form the bulges and retractions assumed by a dehiscing capsule. This example of an early dehiscence stage reproduces the near-ideal form that a tech- nical system would assume under the same circumstances. Living material normally responds differently, less ideal, if this expression is applicable; the lid of the following capsule (Figs. 18a, b) may serve as an example. The original to Fig. 18a, which is illustrated as Fig. 15a,

is seen to bulge as usual in two plaees, tile bigger of the two projections being about three times longer than the smaller one. The latter evolves under an angle of 110 ~ (not 180~ to the bigger.

The present series of pictures and the follo~ing (Figs. 19a-c) reveal the capsule's failure to split gradually in both directions. "Zipping" occurs rather stepwise, usually involving alternate sides. For example: The initial projection (Fig. 18a) pops up a little to the left of the north- south axis. The slit extends at the same time toward the opposite side (arrow) beyond this axis to give rise to another prominent projection there. This stage was not included in Fig. 15 for lack of space. Pro- jection (see Fig. 18b) remains visible on the right-hartd side but is soon

Dehiscence of the Moss Capsule III I i i

Figs. 15 a n d 16. Sequen t iM stages of two dehisc ing capsules. B o t h series p rove t h a t a r t i f ac t s f rom f reeze-dry ing are in generM negl igeable . Fig. 15a-c. The f i rs t a n d las t p i c tu re of th i s series were used to t r ace Figs. 18 a a n d b respec t ive ly . Fig. 16a-e . A second l igh t -op t ica l sequence f rom an- o t h e r capsule. S tage a was en l a rged to get Fig. 19a a n d s tage c reap-

pears as Fig. 19b, whi le Fig. 19c was t a k e n f rom s tage d

112 K. MAIEP,, :

rendered inconspicuous. Projection is forming in the meantime on the left-hand side and finally would have burst out at the opposite side, etc.

The same sequence is displayed by Figs. 16a-d; Figs. 19a-c. Stage a of this second series appears slightly ahead of stage a of the

former and so is stage c with respect to the corresponding stage of the first example. They all could easily be passed off for sequential dehiscence stages of one capsule, were it not for the lid diameters and the slit lengths.

Stage b. The slit has encircled about �89 of the lid circumference (Fig. 9). This side becomes distinctly deformed, while the unopened part preserves more or less the original circular shape.

Fig. 19b gives evidence of a radical change of the appearance of this capsule, definitely related to the length of the slit. The loose flanks are seen to bulge out and at least the lid itself, and possibly also the urn mouth stretch east-west; the conjugated north-south axis is the shortest of all possible diameters. The southern section of this figure shows that the lid margin was actually retracted as was postulated in the previous chapter on the basis of the debatable stage i of Fig. 6 and illustrated by fig. 3. The radius "i" may indicate the approximate position of this stage on the lid margin.

The new lid form evolved on a capsule whose two slit fronts had approached to within 1/s of the lid circumference. This splitting, no doubt, had introduced the instability that ultimately led to the described deformation of the entire distal end of the sporangium.

Stage c. The dehiscence is nearing completion; again the lid has changed shape. Now it appears cordate (Figs. l l and i9c). The "heart" lobes correspond x~dth the two projections now very close together; the notch is the short length of undehisced capsule tissue between both (Fig. l 1--lower left corner), the "heart" tip finally coincides with the slightly transversely compressed lower half of the lid. The lid eventually regains its circular form upon completion of dehiscence.

The urn also undergoes distortion during dehiscence, and in a way sometimes more informative than does the lid. The deformations of

Fig. 17. This figure shows a capsule which breaks up the "ideal" way; this early stage may be compared with Fig. 18a. The separating anulus which causes this oval pattern is seen protruding at the lower section of the lid eireumferenee.~Fig. 18. Stages a and b show the alternating elonga- tion of the slit.~Fig. 19. Comparatively insignificant deformation is dis- played by the first stage a; the slit is still short. But with its length in- creasing to ~ of the capsule circumference and over, the statics of the entire sporangium is getting upset and the form of the capsule changes

aeeordingly: see stages b and c

Dehiscence of the Moss Capsule I I I 113

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Fig. 18

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Fig. 17 0sterr . Bot. Z., Bd. 122, I-I. 1 - -2

Fig. 19 C

l l 4 K. MAIER: Dehiscence of the Moss Capsule I I I

the urn will be described separately and in detail in another paper which is to appear soon.

For their generous help in various ways I am most grateful to Dr. GEOFFreY HAGGIS, Cell Biology Research Insti tute, Depar tment of Agriculture in Ottawa, and to Mr. JAy GEORGE FRAJKO~, Montreal.

Summary This preliminary report presents evidence of characteristic deforma-

tion patterns arising on the lid during dehiscence. These patterns are caused by stresses being set up in the capsule tissue by the detaching anulus. The degree to which the capsule is deformed, depends on the length of the slit between lid and anulus. The splitting in general seems to severely disturb the static equilibrium of the sporangium.

References

MAIER, K., 1967: Zur Dehiszenz der Laubmooskapsel. I. Die Abl6sung des Anulus v o n d e r Kapsel; dargestellt an Funaria hygrometrica L., Osterr. Bot. Z. 114, 51--65.

- - 1973: Dehiscence of the Moss Capsule. II . The anulus--analysis of its functional apparatus. 0sterr. Bet. Z. 122, 75--98.

Address of the author: Dr. K~raT MAIER, MeGill University, Dept. of Biology, P.O. Box 6070, Montreal i01, Que., Canada.