Development of the leafy shoot in Sphagnum (Bryophyta) involves the activity of both apical and...

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New Phytol. (1998), 140, 581–595

Development of the leafy shoot in

Sphagnum (Bryophyta) involves the activity

of both apical and subapical meristems

B ROBERTO LIGRONE" JEFFREY G. DUCKETT#,*

"Facolta[ di Scienze Ambientali, Seconda Universita[ di Napoli, via Arena 22, 81100

Caserta, Italy

#School of Biological Sciences, Queen Mary & Westfield College, Mile End Road,

London E1 4NS, UK

(Received 1 August 1998; accepted 19 August 1998)

This light- and electron-microscope study of four species of Sphagnum reveals that stem elongation involves

meristematic activities unique to the group and hitherto unrecognized. The internal tissue of the mature stem

arises by the concerted activity of an apical (primary) and a subapical (secondary) meristem. The primary

meristem comprises the immediate derivatives of the single apical cell. Following a small number of divisions, the

primary derivatives differentiate into highly vacuolate parenchymatous cells with a storied arrangement.

Subsequently, the large vacuoles are replaced by numerous small vacuoles and the cells then divide repeatedly, by

transverse septa, producing files of about nine short cells. Finally, ninefold elongation of these secondary cells is

responsible for extension growth of the main stem below the mature capitulum. An early step in primary

differentiation is the confinement of pre-existing plasmodesmata to distinct pitted areas. Further enlargement of

the cells during primary and secondary differentiation involves the thickening of non-pitted wall areas, followed

by expansion and thinning out, while the pitted areas remain virtually unchanged. A cortical array of microtubules

is regularly found in association with non-pitted wall areas, while the unexpanded pitted areas are associated with

smooth endoplasmic reticulum showing continuity with desmotubules. Though sharing much the same cytology

as the conducting cells in bryoid mosses, in terms of their development the central stem cells in Sphagnum are not

homologous with those of other mosses. The unique mode of stem development may be an important factor in the

ecological success of Sphagnum.

Key words: Cortical microtubules, differential wall expansion, pitted walls, plasmodesmata, Sphagnum, secondary

meristem.

The genus Sphagnum, the bog mosses, is widespread

in the world, being found on five continents at most

latitudes, with approx. 150–200 species (Gignac,

1993). Sphagnum is a major component of peatland

vegetation and considered in terms of its total mass,

live and dead, it must be regarded as one of the most

important of all plants (Clymo & Hayward, 1982).

Among the reasons for its remarkable biological

success are the ability to colonize oligotrophic wet

habitats and the ability to maintain a low pH that

prevents the growth of most other plants (Clymo &

Hayward, 1982; Clymo, 1984, 1987).

Sphagnum plants consist of leafy shoots of in-

determinate growth with a single apical cell. The

main shoot bears numerous lateral branches of

* To whom correspondence should be addressed.

E-mail : j.g.duckett!qmw.ac.uk

determinate growth arranged in groups (fascicles)

along the stem (Schimper, 1858; Cavers, 1911;

Smith, 1955). The young branches and leaves form

a compact hemispherical head, the capitulum,

around the apex. The growth pattern of lateral

branches varies according to the species and so does

the frequency by which the developing capitulum

forks to form two separate shoots of indeterminate

growth.

As a consequence of their unlimited apical growth

and repeated forking, the older parts of Sphagnum

plants become highly shaded and eventually die. The

only parts remaining alive at some distance from the

apex are groups of cortical cells in the main stem and

the branches, which are able to regenerate new

plants when several-year-old peat cores are exposed

to the light (Clymo & Duckett, 1986). Acropetal

translocation of nutrients from the older parts of the

shoot to the capitulum has been demonstrated with


582 R. Ligrone and J. G. Duckett

the use of radioactive tracers and interpreted as

evidence that Sphagnum has the ability to relocate

solutes internally (Rydin & Clymo, 1989).

The mature stems of the leafy shoots of Sphagnum

comprise a cortical sheath of one-to-several layers of

dead hyaline cells, with one or more layers of cells

with thick walls (sterome) beneath and an internal

parenchymatous cylinder with no intercellular

spaces. Until very recently, the internal cylinder was

thought to show no cytological differentiation

(Schimper, 1858; Cavers, 1911). However, a recent

study (Ligrone & Duckett, 1998) has revealed that

the internal cells of the mature stem have a highly

specialized cytoplasmic organization including an

axial system of microtubules associated with the

nucleus and a range of organelles.

In the present paper we report a further suite of

features associated with the differentiation of the

leafy stems. These are unique to Sphagnum and have

remained unrecognized hitherto. We describe how

stem development occurs in two distinct phases: an

apical (primary) phase, involving differentiation of

derivatives of the apical cell within the capitulum;

and a wave of subapical (secondary) meristematic

activity. The latter is mainly responsible for stem

elongation below the capitulum. This account details

the cytological changes associated with the primary

and secondary meristems, with particular emphasis

on changes in the cell walls.

Living specimens of Sphagnum cuspidatum Ehrh., S.

palustre L., S. recurvum P. Beauv. and S. magel-

lanicum Bridel were collected from several field sites

in southern England during the spring and early

summer. The capitula were isolated and, after

removing the external leaves and branches, were

fixed in a mixture of 2% glutaraldehyde, 1%

formaldehyde (freshly prepared from paraformal-

dehyde) and 0±5% tannic acid in 0±05 Na-phosphate

buffer at pH 7. After 2 h at room temperature under

gentle vacuum the material was rinsed in 0±1 Na-

phosphate buffer and post-fixed overnight with 1%

osmium tetroxide in 0±1 Na-phosphate buffer,

pH 6±8, at 4 °C. The material was then dehydrated in

ethanol, transferred to propylene oxide and slowly

infiltrated with Spurr’s resin at 4 °C (Ligrone &

Duckett, 1994).

Thin sections, cut with a diamond knife, were

stained with 5% uranyl acetate in methanol for

15 min followed by Reynold’s lead citrate for 10 min

and observed with a Jeol 1200 EX2 or a Philips

CM12 electron microscope. One-µm-thick sections

were cut with glass knives, stained with 1% toluidine

blue in borax and photographed with a Leitz

Ortholux microscope.

A morphometric analysis was performed in S.

cuspidatum at seven developmental stages (Table 1).

Table

1.C

hanges

inth

ein

tern

alce

lls

of

the

leafy

stem

duri

ng

pri

mary

(sta

ges

1–4)

and

seco

ndary

(sta

ges

5–7)

diff

eren

tiation

inSphagnum

cusp

idatu

m

Develo

pm

enta

lst

age

Mic

rogra

phs

(Fig

ure

s)

Dia

gra

mm

atic

repre

senta

tions

(Fig

ure

s)C

ell

length

(µm

)C

ell

wid

th(µ

m)

Wall

thic

kness

inpitte

dare

as

(µm

)

Wall

thic

kness

innon-p

itte

dare

as

(µm

)L

ength

of

pitte

dare

as

(µm

)

Pla

smodesm

al

frequency

inlo

ngitudin

al

walls†

11a,2b

7a,9a

25³

48³

20±1

8³

0±0

3*

0±1

8³

0±0

3*

26±3

³6±4

22c–

e7b,9b

43³

610³

30±2

1³

0±0

50±2

8³

0±0

42±0

8³

0±5

615±6

³4±2

32d,g

7c,

9c

55³

613³

30±5

2³

0±0

61±1

2³

0±2

02±4

0³

0±7

012±5

³4±4

43a,b

7d,9d

75³

816³

20±3

9³

0±0

60±5

3³

0±0

82±3

7³

0±8

18±6

³2±6

54

7e,

8a,9e

78³

12

17³

20±4

3³

0±1

21±0

5³

0±2

02±5

0³

0±5

18±8

³1±9

65

7f–

i,8b–d,9f,

g20³

321³

40±6

7³

0±1

52±0

7³

0±5

02±4

1³

0±7

44±1

³2±2

76

7j,

8e,

9h

180³

45

21³

40±8

5³

0±1

80±8

0³

0±1

22±7

1³

0±8

30±7

³0±3

The

num

bers

are

means³

fr

om

at

least

25

measu

rem

ents

.

*N

ocle

ar

cut

dis

tinction

isvis

ible

at

this

stage

betw

een

pitte

dand

non-p

itte

dare

as.

†N

um

ber

of

pla

smodesm

ata

per

10

µm

wall

pro

file

inm

edia

nlo

ngitudin

alse

ctions

of

cells.


Meristems and shoot development in Sphagnum 583

(a)(a)(a) (((ccc))) (((eee)))

(((bbb)))

(((ddd))) (((fff )))

Figure 1. Light microscopy of leafy stem development in Sphagnum. (a) Shoot apex of S. magellanicum,

showing the apical cell (ac) and its immediate derivatives. (b–d ) Longitudinal sections of the shoot apex in S.

magellanicum (b) and S. cuspidatum (c, d ), showing the region of primary differentiation and the onset of

secondary divisions (arrows in b and d ). (e) Higher magnification showing the highly pitted cells that initiate

secondary divisions (arrows) in S. magellanicum. ( f ) Detail of the subapical secondary meristem in S.

cuspidatum. Scale bars: a and f, 100 µm; b, c and d, 200 µm; e, 50 µm.

For each stage, at least 15 low-power electron

micrographs of median longitudinal sections of cells

were utilized. The nucleus was used as a marker of

sections passing through the median longitudinal

plane of cells. The cellular sizes were measured using

light microscopy.

The four species examined showed much the same

morphological organization and developmental pat-

tern. Therefore, except when indicated, the fol-

lowing description applies to all of them.



(a) (d ) (e )

(b) (c)

(g)

(f )

Figure 2. Initial stages of primary differentiation in the leafy stem of Sphagnum cuspidatum. (a) Stem apex,

showing the single apical cell (ac) and its immediate derivatives. (b) Higher magnification of young derivatives

(stage 1). No distinct pitted and non-pitted areas are visible in the cell walls. Abbreviations: mitochondria (m);

plastids (p). (c–e) Stage 2. (c) Light microscopy. (d ) Pitted (arrows) and non-pitted walls areas are clearly

distinct ; the vacuoles (v) and plastids have enlarged appreciably. Note large grana in the plastids. (e) Detail of

a cell wall, showing non-pitted areas with cortical microtubules (arrows) beneath and pitted areas with

numerous plasmodesmata. ( f, g) Stage 3. ( f ) Light microscopy. (g) Detail showing thickening of non-pitted

areas (arrows) and elongation of the nucleus (n). Scale bars: a, b, d and g, 5 µm; c and f, 50 µm; e, 0±5 µm.

Developmental histology of the capitulum

The capitulum terminates with a conical tip bearing

a single apical cell (Fig. 1a). Each segment derived

from the apical cell divides into an outer and an inner

cell. The outer cell divides again, producing an

upper and a lower cell. Each upper cell gives rise to

a leaf primordium and associated mucilage papillae;

the lower cells generate a meristematic cortex that

remains active up to 2–3 mm from the apical cell



(a) (c) (d ) (e )

(b )

(f )

Figure 3. Stem development in Sphagnum cuspidatum. (a, b) Final step of primary differentiation (stage 4). (a)

Light microscopy. (b) Detail showing a prominent vacuole displacing the nucleus (n) to the cell periphery; non-

pitted wall areas (arrows) are thinner and longer than in the previous stage. (c–f ) Initial stage in the

development of secondary meristematic cells. (c) Light microscopy. (d ) Low-power electron micrograph of

a cell containing numerous small vacuoles (v) with electron-opaque precipitates and the nucleus (n), which has

moved back to an internal position; pitted and non-pitted areas in the walls are of similar thickness. (e, f ) Cell

wall details ; the wall material in non-pitted areas is less opaque to electrons and shows a more compact fibrillar

texture than in non-pitted areas; arrows indicate cortical microtubules. Scale bars: a and c, 200 µm; b and d,

5 µm; e, 1 µm; f, 0±5 µm.



(a) (b) (c )

(d )

(f )(e )

Figure 4. Fully developed secondary meristematic cells in the leafy stem of Sphagnum (stage 5). (a, b) Light

microscopy in S. recurvum (a) and S. cuspidatum (b) ; note the heavily stained vacuoles (arrows). Abbreviations:

n, nucleus. (c, e) Details from S. palustre (c) and S. recurvum (e) ; the non-pitted wall areas (arrows) are again

thickened; the vacuoles (v) have become smaller and more numerous. (d ) Tangential section of a longitudinal

wall in S. magellanicum, showing a row of six pitted areas with a high concentration of plasmodesmata. ( f ) High

magnification of a pitted area in S. cuspidatum, showing a cortical network of smooth ER continuous with

desmotubules. Scale bars: a and b, 200 µm; c, 5 µm; d and e, 2 µm; f, 0±5 µm.



(a) (b) (c) (d )

(e) (f )

Figure 5. Details of the subapical secondary meristem in the leafy stem of Sphagnum. (a) Light microscopy

showing the first division (arrow) in a secondary meristem cell of S. cuspidatum. (b) Dividing secondary

meristem cells in S. recurvum. (c) Files of cells at the beginning of secondary differentiation in S. palustre.Primary septa between non-sister cells are arrowed. (d ) Dividing cell showing a well-developed phragmoplast

(arrows) between the two daughter nuclei (n) ; plastids (p) are associated with each nucleus. (e) Newly formed

secondary cell at the end of a cell file in S. cuspidatum, showing both a primary (1) and a secondary (2) septum.

( f ) Detail of a cell wall in S. magellanicum, showing cortical microtubules (arrows) along a non-pitted area but

lacking at the level of the pitted area nearby. Scale bar: a, 20 µm; b, 100 µm; c, 200 µm; d and e, 5 µm; f, 0±5 µm.

(Fig. 1b, c) and produces the lateral branches and the

hyaline tissue ensheathing the mature stem.

The inner derivatives of the apical cell generate

the internal cylinder of the stem through a two-step

process. During the first step, which we refer to as

primary differentiation, the cells stop dividing after

a few divisions, become highly vacuolate and enlarge

(Fig. 1b, c). At a distance of 0±7–1±2 mm from the

apical cell the cells form thick, pitted walls and their

cytoplasm becomes denser (Fig. 1b,d, e). Following

these changes, the cells divide repeatedly by trans-

verse septa (Fig. 1e), thus forming a subapical

meristem about 0±8–1±5 mm in length (Fig. 1 f ), the

derivatives of which will generate the internal

cylinder of the mature stem by secondary differen-

tiation (Figs 2–6).

The following description of cytological events

accompanying primary and secondary differentiation

focusses on the internal cells of the cylinder, which

form the bulk of the mature stem. Morphological

and cytological differences between these and the

thick-walled peripheral cells of the cylinder arise

mostly during secondary differentiation and have

been described in a previous study (Ligrone &



(a) (b) (d ) (f )

(c) (g) (h)

Figure 6. Secondary differentiation of leafy stem cells in Sphagnum. (a) Light microscopy of the mature stem

of S. palustre. The external sheath of hyaline cells (arrows) arise from the cortical meristem; all the internal

tissue, including the stereids, is of secondary origin. (b) Detail from a showing fully elongate internal cells. (c)Detail of an internal cell in the course of elongation in S. cuspidatum. The non-pitted wall areas are very thin

and long, but the pitted area (arrow) retains much the same length and thickness as in former stages; n, nucleus.

(d–f ) Details of the cell wall in the course of (d, e) and at the end of ( f ), secondary expansion in S. cuspidatum.

Associated with the expanding wall are numerous cortical microtubules (arrowed) perpendicular to the cell



Duckett, 1998). Quantitative parameters of cellular

differentiation in S. cuspidatum are summarized in

Table 1 and the developmental changes illustrated

diagrammatically in Figures 7–9. For clarity we have

divided development into 7 stages: stages 1–4 refer

to primary differentiation of the inner derivatives of

the apical cell ; stages 5–7 refer to secondary

differentiation of the inner cells of the stem.

Primary differentiation

The apical cell has dense cytoplasm with a prominent

spheroidal nucleus, numerous small plastids and

vacuoles and, frequently, dumbell-shaped mito-

chondria (Fig. 2a). The immediate derivatives have

a similar ultrastructural morphology, except for the

vacuoles, which are more irregular in shape and

often accumulate electron-opaque material (Fig. 2b).

At this stage (stage 1) the cells present slightly

elongate hexagonal longitudinal profiles. They

usually have very thin walls (about 0±15–0±20 µm)

with numerous evenly scattered plasmodesmata. The

transition to stage 2 (i.e. the onset of primary

differentiation) is characterized by cell elongation

(Table 1) with the cells developing a clearly storied

(tiered) arrangement (Fig. 1c, d ) that persists until

the onset of secondary differentiation. The vacuoles

enlarge (Fig. 2c, d ) as do the plastids, which also

develop an extensive inner-membrane system (Fig.

2d ). The plasmodesmata in longitudinal walls now

become confined to distinct pitted regions separated

from each other by non-pitted areas (Fig. 2d ). The

transverse walls usually contain a single, central,

pitted area and a relatively small margin with no

plasmodesmata. From this stage, a cortical array of

microtubules with a transverse orientation is regu-

larly found along the non-pitted wall areas, while no

microtubules are visible in the cytoplasm adjoining

pitted regions (Fig. 2e). A collar of electron-

transparent material surrounds each plasmodesma;

moreover, the wall material between plasmodesmata

in the same pitted area is more electron-opaque than

in adjacent non-pitted areas (Fig. 2e).

The subsequent stage in primary differentiation

(stage 3) comprises a change in nuclear shape from

spheroidal to elongate, and a marked thickening of

non-pitted wall areas along with a moderate increase

in cell size (Table 1; Fig. 2 f, g). In stage 4, the cells

elongate considerably, and the numerous vacuoles

present in stage 3 are replaced with one or a few large

vacuoles that displace the nucleus and most

organelles to the periphery (Fig. 3a, b). Cellular

elongation only affects the non-pitted wall areas,

which thus become longer and thinner, whereas the

axis ; the last-deposited wall layers exhibit an axial orientation of cellulose microfibrils ( f, arrows). (g) Mature

internal cell showing secondary wall layering (arrows) in S. recurvum ; pw, primary wall. (h) Detail from g ;

secondary wall layering is conspicuous in the longitudinal walls but it is very scarce on the transverse septa.

Scale bars: a, 200 µm; b, 50 µm; c, d, f and g, 5 µm; e, 0±5 µm; h, 1±0 µm.

pitted areas retain much the same dimensions (Fig. 3

b, Table 1). The same is observed in transverse walls,

where expansion only affects the non-pitted area.

Electron-opaque finely granular material is now

visible in the vacuoles (Fig. 3b). At the end of

primary differentiation, the cells appear as highly

vacuolate parenchyma cells. An approximate evalu-

ation of changes in cell wall volumes indicates that a

very limited deposition of new wall material occurs

during the transition from stage 3 to stage 4.

Although quite variable, the region of primary

differentiation is markedly longer in S. cuspidatum

than in the other three species (cf. Fig. 1b, c). In

S. palustre, cellular vacuolization is more pro-

nounced throughout development and the different

stages in primary differentiation are not so distinct as

in the other species. By contrast, the pattern of

secondary development is remarkably uniform in all

the species examined.

The development of secondary meristematic activity

The first signs of the development of a secondary

meristem from fully differentiated primary cells are

the replacement of the large vacuole(s) by numerous

smaller vacuoles containing electron-opaque coarse

deposits, and the concomitant migration of the nucleus

from the periphery to the interior of the cells (Fig.

3c, d ). Initially, the non-pitted areas are only slightly

thicker than pitted areas, although morphological

differences between the two regions are still evident

(Fig. 3e, f ). Subsequently, the non-pitted wall areas

become strongly thickened while retaining a homo-

geneous fibrillar structure with no visible layering.

The vacuoles further increase in number and reduce

in size (stage 5; Fig. 4a–c, e). Each cell is usually in

contact laterally with five or six other cells and there

are rows of six to eight pitted areas on each cellular

face (Fig. 8). The pitted areas are elliptical or

roughly circular in shape, with a diameter of

about 2–6 µm, and contain numerous plasmodesmata

(Fig. 4d ) with a density ranging from 22 µm−#

(S. magellanicum and S. recurvum) to 30 µm−# (S.

palustre and S. cuspidatum). Plasmodesma numbers

per pit thus range from 60 to 800 with most contain-

ing 200–500. A network of tubular endoplasmic

reticulum (ER), showing continuity with the desmo-

tubules, is regularly found in the cortical cytoplasm

adjoining pitted areas (Fig. 4 f ).

The cells reach their maximum width just before

the onset of the secondary divisions. The first of

these separates two cells of either similar or different

sizes (cf. Figs 1d, e and 5a). Irrespective of whether

the first division is symmetrical or asymmetrical, this



(a)

n

vn

(b ) (c )

v

(d )

n

v

(e )

n

mt

v

(f )

n

n

mt

v

n

(g ) (h ) ( j )

(i )

v

mt

n

Figure 7. For legend see opposite.



is followed during stage 6 by other, both terminal

and intercalary, divisions (Fig. 5b) ; these produce

files of 6–11 (occasionally more but on average nine)

short cells (Fig. 5c). A tightly controlled balance of

cellular elongation and cellular division keeps the

initial length of secondary derivatives rather constant

(about 20 µm in S. cuspidatum, slightly more in the

other species). The files of newly formed daughter

cells are conspicuously longer than the mother cells

from which they arise (about 180 µm as compared

with about 80 µm in S. cuspidatum). In spite of this

considerable expansion, the longitudinal walls are

further thickened in the course of the divisions (Fig

5a, b, e). As in former stages, the thickening only

affects non-pitted areas, which exhibit a compact

fibrillar texture with a predominately transverse

orientation of microfibrils (Fig. 5 f ). Cortical micro-

tubules in the adjoining cytoplasm also show trans-

verse orientation (Fig. 5 f ). Cellular division is

preceded by plastid migration to the nuclear poles,

probably ensuring that the daughter cells receive

equivalent plastid numbers, and terminates with a

phragmoplast depositing a new septum between the

two daughter nuclei (Fig. 5d ). The new transverse

walls are slightly thinner and less electron-opaque

than the old ones; moreover, they lack the non-

pitted, thickened margin present in the transverse

septa of primary origin (Fig. 5e).

Secondary differentiation

During subsequent differentiation, the secondarily

formed cells elongate about ninefold while remain-

ing almost unchanged in width (Fig. 6a, b). The

longitudinal walls of the elongating cells are very

thin (Fig. 6c), show a loose fibrillar texture with a

predominately transverse or oblique orientation and

have numerous cortical microtubules beneath (Fig.

6d, e). In the fully elongated cells, the longitudinal

walls are slightly thicker because of the late de-

position of additional layers with a lengthwise

orientation of microfibrils (Fig. 6 f ). Once again, the

elongation does not affect the pitted areas; these

retain much the same length and morphology that

was established at stage 2 (Fig. 6c and Table 1). An

evaluation of cell wall volumes in unexpanded and

Figure 7. Diagrammatic representation of the major subcellular changes in the ontogeny of the central cells

of Sphagnum stems. Abbreviations; mt, microtubules; n, nuclei ; v, vacuoles. Because of economies of scale,

plastids, mitochondria, ER, dictyosomes and plasmodesmata are not illustrated. Figure numbers in parentheses

refer to micrographs of the various stages. All drawings are to the same scale. (a) Stage 1: thin-walled

rectangular cells (Fig. 2b). (b) Stage 2: elongation of the initially hexagonal apical cell derivatives has produced

a storied arrangement of elongate cells with large vacuoles (Fig. 2c, d ). (c) Stage 3: further cell enlargement is

associated with wall thickening, nuclear elongation and a reduction in the size of the vacuoles (Fig. 2 f, g). (d )

Stage 4: note the now peripheral nucleus, thinner walls and large central vacuole (Fig. 3a, b). (e) Stage 5:

Secondary meristematic cell with thick pitted walls, an elongate central nucleus and small vacuoles with dense

contents (Fig. 4). ( f–i) Stage 6: the secondary meristematic cells undergo several divisions to produce files of

6-9-11 cells (Fig. 5) concomitant with cell elongation and thinning of the walls. These processes continue after

the cessation of meristematic activity (i), when axial endoplasmic strands begin to appear. ( j) Stage 7: mature

central cell with endoplasmic strands, polarized cytoplasm and thin walls (Fig. 6).

(a ) (b ) (d ) (e )

(c )

Figure 8. Diagrammatic representation showing the

apportioning of pits in the secondary meristematic cells

during stages 6 and 7. Numbers in parentheses refer to

micrographs and to the drawings of the same stages in

Fig. 7. (a) Secondary meristematic cell with a row of pits

on each longitudinal face (Figs 7e ; 4c–e). (b) The first

division of the meristematic cells halves the number of pits

on each face (Figs 7 f, 5a). (c) Subsequent divisions again

halve the number of pits per face (Fig. 7g). (d ) Following

the final division each face contains but one pit (Figs 7h,

6c). (e) The single pit in each face remains unchanged

during maturation of the central cells (Fig. 7 j).

fully elongated cells of S. cuspidatum indicates that

cellular elongation during secondary differentiation

must involve the synthesis of approx. 200% new

wall material. Further thickening of the longitudinal

walls follows the completion of cellular expansion in

S. palustre, S. recurvum and S. magellanicum (Fig.

6g,h) ; no secondary layering is usually visible in S.

cuspidatum (Fig. 6c). A full description of the

cytology of the mature cells is given in Ligrone &

Duckett (1998).



(h)(g)(f )(e )(d )(c )(b )(a )

2 lm(a )

2 lm(b ) – (h )

Figure 9. Diagrammatic representation of the changes in wall thickness, pit spacing and cortical microtubule

distribution during central cell ontogeny in Sphagnum. Numbers in parentheses refer to micrographs and to the

drawings of the same stages in Figure 7. (a) Apical cell derivative; thin wall with uniformly distributed

plasmodesmata and microtubules (Figs 7a, 2a, b). (b) Stage 2: distinct pitted and non-pitted areas (Figs 7b, 2d,

e). Shading with arrows indicates the wall segments that have expanded between stages a and b. (c) Stage 3:

thicker walls between the pitted areas (Figs 7c, 2g). (d ). Stage 4: thinner walls and wider areas between the pits

(Figs 7d, 3b). (e) Stage 5: secondary meristematic cell with thicker walls (Figs 7e, 4). ( f ) Onset of stage 6; the

walls reach their maximum thickness and the distance between the pits remains the same as in stage 4 (Figs 7 f,5e, f ). (g) Stage 6: midway; elongation of the derivatives of the secondary meristematic cells accompanied by

an increased spacing of the pits (Fig. 7 i). (h) Stage 7: mature central cell ; the thin walls now generally contain

no more than one pit per lateral face (Figs 7 j, 6c).

This study shows that stem development in

Sphagnum is a two-step process. Primary meris-

tematic activity in derivatives of the apical cell

produces tiers of differentiated, highly vacuolate

cells in the centre of the capitulum. In the secondary,

subapical phase, redifferentiation and resumption of

meristematic activity in these cells, producing files of

elongate derivatives, is responsible for stem

elongation underlying the expansion of the mature

capitulum.

Though the presence of subapical meristematic

cells, dividing transversely, can be surmised from

previously published drawings of Sphagnum capitula

by Schimper (1858), a subapical secondary meristem

has not previously been reported. Moreover, this

appears to be a feature unique to Sphagnum not only

among the mosses (cf. Crosby, 1980) but also among

all bryophytes. Examples of intercalary meristems

are known in other bryophytes: in the young

sporophyte of mosses (with the exception of

Sphagnum) (Roth, 1969; Crandall-Stotler, 1981,

1984); in the young gametangiophores of Reboulia

(Brown & Lemmon, 1990) and other marchantialean

liverworts (Marchantia and Preissia Cavers, 1904;

Goebel, 1905); and notably in the sporophyte of

anthocerotes (Crandall-Stotler, 1981). These, how-

ever, are instances of residual meristems of primary

origin rather than true secondary meristems.

In an evolutionary context, although at maturity

the central stem cells of Sphagnum cytologically

closely resemble the food-conducting cells of bryoid

mosses (Ligrone & Duckett, 1994), developmentally

they are not homologous. The transverse end walls

in Sphagnum clearly relate to the secondary origin of



these cells, whereas the inclined end walls in bryoid

conducting cells reflect their primary development

directly from derivatives of the apical cell.

On average, each secondary initial in Sphagnum

produces nine derivatives, each elongating to about

180 µm, with a resulting overall length of about

1600 µm for each file of cells. This accounts for the

considerable extension of the main stem recorded for

several Sphagnum spp. (Clymo, 1970; Sonesson et

al., 1980; Clymo & Hayward, 1982; Gerdol, Bonora

& Poli, 1994). By comparison, in the setae of

jungermannialean and metzgerialean liverworts,

which typically expand from 1 to 35–50 mm in a few

hours or days, elongation is caused solely by

vacuolation of individual cells with no cell divisions

(Thomas & Doyle, 1976; Schnepf & Deichgra$ ber,

1979). Single epidermal cells in the seta of Pellia

grow from 12 to about 600 µm in length within 24 h

(Schnepf & Deichgra$ ber, 1979); this figure, although

large, is much below the single-cell increment

achieved in Sphagnum by interposition of secondary

meristem activity.

Although this study focuses on only four species,

representing but two sections in the genus

Sphagnum, less-detailed observations on a range of

other species (Ligrone & Duckett, 1998) indicate

that the presence of a subapical secondary meristem

is ubiquitous in the genus. This unusual growth

mechanism is probably closely associated with the

equally unusual ecology of bog mosses and their

success in waterlogged oligotrophic habitats.

Sphagnum plants frequently keep growing through-

out the year, notably at temperate latitudes, although

generally the bulk of the growth occurs during

summer (Clymo, 1970; Clymo & Hayward, 1982).

The secondary meristem activity ensures that the

capitula, the most important part of the plants in

terms of photosynthetic activity (Johansson &

Linder, 1990), are kept at the surface; this has

benefits in terms of light interception and CO#

uptake. In addition, the secondary meristem might

be crucial for long-term maintenance of the growth

pattern of Sphagnum, because it permits rapid

elongation of old capitula when these are being

replaced by new ones after periods of dormancy

caused, for example, by winter freezing or summer

droughts. In striking contrast, other large perennial

mosses that grow in dense tufts (e.g. many members

of the Polytrichales), but which do not concentrate

their photosynthetic tissues at the stem apices, lack a

secondary meristem (He!bant, 1977).

A second remarkable aspect of leafy stem growth

in Sphagnum is the peculiar way the cells elongate.

At the onset of primary differentiation, moderate

cellular enlargement results in the formation of

distinct pitted areas. The concomitant decrease in

plasmodesmal frequency (Table 1) is fully consistent

with a dilution effect of cell elongation, suggesting

that the development of pitted areas involves the

confinement of pre-existing plasmodesmata rather

than the obliteration of old plasmodesmata and the

formation of new ones. Further cellular growth,

during both primary and secondary differentiation,

involves thickening of non-pitted areas followed by

expansion and thinning out. By contrast, the pitted

areas only undergo very limited thickening and

remain virtually unaltered in size. This is a re-

markable instance of differential expansion of ad-

jacent wall areas in the same cell.

According to our evaluation, the expansion and

thinning-out of pre-thickened wall areas can account

completely for cellular elongation during the tran-

sition from stage 3 to stage 4, with no need for

concomitant synthesis of new wall material. By

contrast, the same mechanism can be responsible for

only about 27% of cellular elongation during

secondary differentiation. The expansion of pre-

thickened wall areas is restricted to the initial phase

of secondary differentiation, resulting in the

formation of very thin uniform walls whose further

elongation must require the synthesis of new wall

material. Like longitudinal growth, the cellular

growth in diameter also depends on the expansion of

the non-pitted area in transverse walls and is

therefore restricted to primary differentiation, be-

cause secondarily formed septa apparently lack a rim

of expandable wall.

As in Sphagnum, cellular expansion in the seta of

Pellia also involves the initial thinning out of pre-

thickened walls, followed by an expansion phase

entirely sustained by the synthesis of new wall

material (Schnepf & Deichgra$ ber, 1979). In Pellia,

however, the process of wall thickening and ex-

pansion is not restricted to specific areas, but instead

affects all the whole longitudinal walls. In both

systems, the pre-thickened walls have a pre-

dominately transverse orientation of microfibrils,

with no visible layering, and are consistently

associated with cortical microtubules showing the

same orientation as the microfibrils.

The plasmodesmal frequency decreases linearly

with cellular elongation during the whole devel-

opmental sequence in stem cells of S. cuspidatum,

indicating that at no stage in differentiation do new

plasmodesmata arise. The same is true during

elongation of the water-conducting cells in the

liverwort Symphyogyna (Ligrone & Duckett, 1996).

By contrast, an analysis of plasmodesmal frequency

in expanding leaflet cells of S. palustre supports de

novo formation of plasmodesmata (Schnepf & Sych,

1983).

A major difference between pitted and non-pitted

areas is the absence of microtubules (MTs) in the

cortical cytoplasm adjoining the former. Here the

MTs are replaced with a network of tubular ER

continuous with the desmotubules. A similar con-

dition was recently reported in developing water-

conducting cells of the liverwort Symphyogyna,



where the dense array of cortical MTs associated

with the longitudinal walls avoids the wall areas

surrounding single plasmodesmata; these areas also

exhibit different staining properties from the rest

of the wall (Ligrone & Duckett, 1996). In

Symphyogyna, however, local differences in the wall

structure arise along with the deposition of a

secondary wall (i.e. after the completion of cellular

elongation) and affect the differential lysis of the wall

after the death of the cells rather than wall expansion.

This is not the case in Sphagnum, where pitted and

non-pitted areas are both part of the primary wall

and clearly recognizable since a very early de-

velopmental stage. The different thickening proper-

ties of pitted and non-pitted areas are presumably

linked to the uneven distribution of MTs and cortical

ER and probably reflect a differential control of

cellulose deposition and of wall-loosening and wall-

stiffening processes (Cosgrove, 1993a, b ; Cyr, 1994).

Apart from tip-growing protonemal cells (Sievers

& Schnepf, 1981), other precedents for localized cell

wall expansion is intercalary growth in young thallus

cells in Marchantia (Apostolakos & Galatis, 1993)

and in the initial cells of gemmae in jungerman-

nialean liverworts (Duckett & Ligrone, 1995) and the

moss Aulacomnium androgynum (Hedw.) Schwaegr.

(Ligrone, Duckett & Gambardella, 1996b). In con-

trast to Sphagnum, here the expanding wall areas do

not undergo preliminary thickening, although they

are consistently associated with cortical MTs. Also

different from the mechanism observed in Sphagnum

is the process of localized wall expansion in fully

differentiated cells of mosses such as the initials of

foliar gemmae in Tortula (Ligrone, Duckett &

Gambardella, 1996a) or protonemal cells during the

development of lateral branches (Schmiedel &

Schnepf, 1979; Ligrone, Duckett & Egunyomi,

1992). Here expansion involves the deposition of a

highly plastic new wall layer and entails breaking off

of the old wall.

The occurrence of highly pitted cells in the leafy

shoot apex of Sphagnum was probably first reported

by Schimper (1858), who assumed that the apparent

absence of pits in cells of mature parts of the stem

resulted from obliteration by new wall material. The

same view was expressed in a more recent study

using scanning electron microscopy (Baker, 1988).

The present study reveals that the pits arise by

differential deposition of primary wall material

during primary differentiation. The pits are then

allocated to separate cells during secondary divisions

and eventually become scarcely visible as a conse-

quence of the expansion and thinning out of the cell

walls during secondary differentiation; however,

they are never obliterated by new wall material. In

striking contrast, primary plasmodesmata and the

adjacent walls are modified profoundly during

phloem differentiation (Glockmann & Kollmann,

1996).

From the functional standpoint, the changes in

plasmodesmal distribution in the stems of Sphagnum

almost certainly reflect changes in the major

symplastic pathways. Lateral transport, involving

the growing branches, probably predominates within

the capitulum, but below the capitulum translocation

will be exclusively axial. Thus, plasmodesmata are

approximately equally distributed between lateral

and end walls in the capitulum, but below the

capitulum those in the end walls outnumber the

laterals by at least one order of magnitude.

R.L. thanks Queen Mary & Westfield College for

laboratory facilities in 1992, 1993 and 1996. The obser-

vations were in part performed at the CIRUB (University

of Naples ‘Federico II’).

Apostolakos P, Galatis B. 1993. Interphase and preprophase

microtubule organization in some polarized cell types of the

liverwort Marchantia paleacea Bert. New Phytologist 124 :

409–421.

Baker RGE. 1988. The morphology and distribution of pits in the

cell walls of Sphagnum. Journal of the Hattori Botanical

Laboratory 64 : 359–365.

Brown RC, Lemmon BE. 1990. Polar organizers mark division

axis prior to preprophase band formation in mitosis of the

hepatic Reboulia hemisphaerica (Bryophyta). Protoplasma 156 :

74–81.

Cavers F. 1904. On the structure and biology of Fegatella conica.

Annals of Botany 18 : 87–119.

Cavers F. 1911. The inter-relationships of the Bryophyta. VI.

Sphagnales. New Phytologist 10 : 1–21.

Clymo RS. 1970. The growth of Sphagnum : methods of

measurement. Journal of Ecology 58 : 13–49.

Clymo RS. 1984. Sphagnum-dominated peat bog: a naturally acid

ecosystem. Philosophical Transactions of the Royal Society of

London B305 : 487–499.

Clymo RS. 1987. Interactions of Sphagnum with water and air.

In: Hutchinson TC, Meema KM, eds. Effects of Atmospheric

Pollutants on Forests, Wetlands and Agricultural Ecosystems.

Berlin, Germany: Springer Verlag, 513–529.

Clymo RS, Duckett JG. 1986. Regeneration of Sphagnum. New

Phytologist 102 : 589–614.

Clymo RS, Hayward PM. 1982. The ecology of Sphagnum. In:

Smith AJE, ed. Bryophyte Ecology. London, UK: Chapman and

Hall, 229–289.

Cosgrove DJ. 1993a. How do plant cell walls extend? Plant

Physiology 102 : 1–6.

Cosgrove DJ. 1993b. Wall extensibility : its nature, measurement

and relationship to plant cell growth. New Phytologist 124 :

1–23.

Crandall-Stotler B. 1981. Morphology}anatomy of hepatics and

anthocerotes. Advances in Bryology 1 : 315–398.

Crandall-Stotler B. 1984. Musci, hepatics and anthocerotes – an

essay on analogues. In: Schuster RM, ed. New Manual of

Bryology, vol. 2. Nichinan, Japan: Hattori Botanical Lab-

oratory, 1093–1129.

Crosby MR. 1980. The diversity and relationships in mosses. In:

Taylor RJ, Leviton AE, eds. The Mosses of North America. San

Francisco CA, USA: Pacific Division, AAAS, 115–129.

Cyr RJ. 1994. Microtubules in plant morphogenesis : role of the

cortical array. Annual Review of Cell Biology 10 : 153–180.

Duckett JG, Ligrone R. 1995. The formation of catenate foliar

gemmae and the origin of oil bodies in the liverwort

Odontoschisma denudatum (Mart.) Dum. (Jungermanniales) : a

light and electron microscope study. Annals of Botany 76 :

405–419.



Gerdol R, Bonora A, Poli F. 1994. The vertical pattern of

pigment concentration in chloroplasts of Sphagnum

capillifolium. Bryologist 97 : 158–161.

Gignac LD. 1993. Distribution of Sphagnum species, com-

munities, and habitats in relation to climate. Advances in

Bryology 5 : 187–222.

Glockman C, Kollmann R. 1996. Structure and development of

cell connections in the phloem of Metasequoia glyptostroboides

needles. I. Ultrastructural aspects of modified primary plasmo-

desmata in Strasburger cells. Protoplasma 193 : 191–203.

Goebel K. 1905. Organography of Plants. Trans. Balfour IB.

Oxford, UK: Clarendon Press.

He! bant C. 1977. The conducting tissues of bryophytes. Bryo-

phtorum Bibliotheca 10 : 1–157.

Johansson LG, Linder S. 1990. Photosynthesis of Sphagnum in

different microhabitats on a subarctic mire. In: Sonesson M,

ed. Ecology of a Subarctic Mire. Stockholm, Sweden: Ecological

Bulletins 30 : 181–190.

Ligrone R, Duckett JG. 1994. Cytoplasmic polarity and

endoplasmic microtubules associated with the nucleus and

organelles are ubiquitous features of food-conducting cells in

bryoid mosses (Bryophyta). New Phytologist 127 : 601–614.

Ligrone R, Duckett JG. 1996. Development of water-conducting

cells in the antipodal liverwort Symphyogyna brasiliensis

(Metzgeriales). New Phytologist 132 : 603–615.

Ligrone R, Duckett JG. 1998. The leafy stems of Sphagnum

(Bryophyta) contain highly differentiated polarized cells with

axial arrays of endoplasmic microtubules. New Phytologist 140 :

567–579.

Ligrone R, Duckett, JG, Egunyomi A. 1992. Foliar and

protonemal gemmae in the tropical moss Calymperes

(Calymperaceae) : an ultrastructural study. Cryptogamic Botany

2 : 317–329.

Ligrone R, Duckett JG, Gambardella R. 1996a. Serial

development of foliar gemmae in Tortula (Pottiales, Musci), an

ultrastructural study. Annals of Botany 78 : 305–315.

Ligrone R, Duckett JG, Gambardella R. 1996b. Development

and liberation of cauline gemmae in the moss Aulacomnium

androgynum (Hedw.) Schwaegr. (Bryales) : an ultrastructural

study. Annals of Botany 78 : 559–568.

Roth D. 1969. Embryo und embryotheca bei den Laubmoosen.

Eine histogenetische und morphologische Untersuchung.

Bibliotheca Botanica 129 : 1–49.

Rydin H, Clymo RS. 1989. Transport of carbon and phos-

phorous compounds about Sphagnum. Proceedings of the Royal

Society of London B237 : 63–84.

Schimper WP. 1858. Versuch einer Entwicklungsgeschichte der

Torfmoose (Sphagnum) und einer Monographie in der Europa

verkommenden Arten dieser Gattung. Stuttgart, Germany:

Schweitzerbart’s Verlagshandlung.

Schmiedel G, Schnepf E. 1979. Side branch formation and

orientation in the caulonema of the moss, Funaria hygrometrica :

normal development and fine structure. Protoplasma 100 :

367–383.

Schnepf E, Sych A. 1983. Distribution of plasmodesmata in

developing Sphagnum leaflets. Protoplasma 116 : 51–56.

Schnepf E, Deichgra$ ber G. 1979. Elongation growth of setae of

Pellia (Bryophyta) : fine structural analysis. Zeitschrift fuX rPflanzenphysiologie 94 : 283–297.

Sievers A, Schnepf E. 1981. Morphogenesis and polarity of

tubular cells with tip growth. In: Kiermayer O, ed. Cyto-

morphogenesis in Plants. Wien, Austria : Springer Verlag,

265–299.

Smith GM. 1955. Cryptogamic Botany. New York, USA:

McGraw-Hill Book Company.

Sonesson M, Persson S, Basilier K, Stenstrøm TA. 1980.Growth of Sphagnum riparium Angstr. In relation to some

environmental factors in the Stordalen mire. In: Sonesson M,

ed. Ecology of a Subarctic Mire. Stockholm, Sweden: Swedish

Natural Science Research Council, 191–207.

Thomas RJ, Doyle WT. 1976. Changes in the carbohydrate

constituents of elongating Lophocolea heterophylla setae

(Hepaticae). American Journal of Botany 63 : 1054–1059.

Development of the leafy shoot in Sphagnum (Bryophyta) involves the activity of both apical and...

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