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Journal of Bryology

ISSN: 0373-6687 (Print) 1743-2820 (Online) Journal homepage: http://www.tandfonline.com/loi/yjbr20

Stomatal differentiation and abnormal stomata in


hornworts
Silvia Pressel, Tomasz Goral & Jeffrey G. Duckett
To cite this article: Silvia Pressel, Tomasz Goral & Jeffrey G. Duckett (2014) Stomatal
differentiation and abnormal stomata in hornworts, Journal of Bryology, 36:2, 87-103
To link to this article: http://dx.doi.org/10.1179/1743282014Y.0000000103

Published online: 15 May 2014.

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Stomatal differentiation and abnormal


stomata in hornworts
Silvia Pressel1, Tomasz Goral2, Jeffrey G. Duckett1
Life Sciences Department, The Natural History Museum, London, UK, 2Imaging and Analysis Centre, The Natural
History Museum, London, UK

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This light and electron microscope study reveals considerable uniformity in hornwort stomata morphology
and density in contrast to common spatial and developmental abnormalities in tracheophytes and mosses.
Stomata arise from a median longitudinal division of sporophyte epidermal cells morphologically
indistinguishable from their neighbours apart from the retention of a single chloroplast whilst those in the
other epidermal cells fragment. Chloroplast division and side-by-side repositioning of the two daughter
chloroplasts determines the division plane in the stomatal mother cell. The nascent guard cells contain
giant, starch-filled chloroplasts which subsequently divide and, post aperture opening, regain their
spherical shape. Accumulation of wall material over the guard cells and of wax rodlets lining the pores
follows opening. While the majority of stomata are bilaterally symmetrical those lining the dehiscence
furrows display dextral and sinistral asymmetry due to differential expansion of the adjacent epidermal
cells.
The ubiquity of open stomata suggests that these never close with the maturational wall changes rendering
movement extremely unlikely. These structural limitations, a liquid-filled stage in the ontogeny of the
intercellular spaces, and spores already at the tetrad stage when stomata open, suggest that their primary
role is facilitating sporophyte desiccation leading to dehiscence and spore dispersal rather than gaseous
exchange. Stomata ontogeny and very low densities, like those in Devonian fossils, suggest either ancient
origins at a time when atmospheric carbon dioxide levels were much greater than today or a function other
than gaseous exchange regulation. We found no evidence for stomatal homology between hornworts,
mosses and tracheophytes.
Keywords: Anthocerotophyta, Capsule dehiscence, Desiccation biology, Guard cells, Stomatal evolution

Introduction
Genetic and hormonal manipulation of stomatal
density in flowering plants (Tal et al., 1970; Martin
& Stimart, 2005; Doheny-Adams et al., 2012; Zhang
et al., 2012), together with investigations into the
effects of carbon dioxide concentrations (McElwain &
Chaloner, 1995; Woodward & Kelly, 1995; Beerling,
2007; Konrad et al., 2008), are at the forefront of
current research on yield improvement and climate
change. Alongside these experimental studies a wide
variety of stomatal defects, affecting both guard cell
and subsidiary cell morphology and vitality, have been
reported from numerous vascular plants (Dehne, 1961;
Takahashi, 1962; Inamdar & Patel, 1969; Inamdar
et al., 1969; Gopal & Shah, 1970; Inamdar et al., 1970;
Inamdar & Patel, 1976).
In contrast, there has never been a thorough
investigation of stomatal abnormalities in bryophytes.

Correspondence to: S Pressel, Life Sciences Department, The Natural


History Museum, Cromwell Road, London, SW7 5BD, UK. Email:
s.pressel@nhm.ac.uk

British Bryological Society 2014


DOI 10.1179/1743282014Y.0000000103

Kuhlbrodt (1922) described several abortive structures


and suggested links between bryophyte stomata and
those in pteridophytes. Haberlandt (1886) and Paton
(1957) reported the regular occurrence of stomata with 3
or 4 guard cells, which they regarded as freaks, in
several groups of mosses. Paton and Pearce (Paton,
1957; Paton & Pearce, 1957) also recorded the occasional
occurrence in mosses of two adjacent stomata at variance
with the one cell spacing rule (Rudall et al., 2013).
In hornworts, Campbell (1895) described the
stomata as arising from epidermal cells that cease
to elongate, become oval in shape and then divide
longitudinally into two guard cells whose median
walls subsequently split to form the pores. Whereas in
many mosses the cells surrounding stomata can be
distinguished from other epidermal cells by their
radial arrangement (Paton, 1957), indicating that
ontogeny depends on patterns of cell divisions
extending beyond the guard cells, recalling many
similar situations in angiosperms (Evert, 2006), in
hornworts only a single epidermal cell is involved and
the adjacent epidermal cells are unaffected.

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Campbell (1895) recorded that the sporophytic


epidermal cells in hornworts contain one large
chloroplast whereas Cavers (1911) reports two as is
the sporophytic norm compared with the monoplastidic gametophyte cells. Other authors described the
deposition of additional wall materials over the
epidermis and guard cells, variously referred to as
cutin or mucilage (Lee & Priestley, 1924; Isaac, 1941;
Smith, 1955) and Paton (1957) remarked that the
pores become filled with cuticle, a feature also found
in the Bryales.
Paton (1957) commented that the stomata in
hornworts are usually widely isolated from each
other and are mostly vertically aligned with the
axes of the sporophytes, though she also noted
two adjacent stomata as well as those with single
guard cells. The presence (in Anthoceros, Folioceros,
Leiosporoceros, Paraphymatoceros, Phaeoceros and
Phaeomegaceros) or absence (in Dendroceros, Megaceros, Nothoceros and Notothylas) of stomata is a
key generic character in hornworts while density
is specifically diagnostic in Folioceros (Asthana &
Srivastava, 1991).
Hornwort stomata have been reported to respond
to environmental stimuli and exogenous abscisic
acid (ABA) (Hartung et al., 1987, 1994; Bopp &
Werner, 1993; Christianson, 2000), a substance also
occurring naturally and associated with desiccationtolerance in the group. However, Lucas & Renzaglia
(2002) found no evidence for stomatal closure in
response to ABA or darkness, with some response
to desiccation. Fast Violet B staining in the guard
cells was associated with pore opening thus suggesting organic acid accumulation (e.g. malate), but
cobaltous nitrate staining for potassium produced
equivocal results between open and closed stomata.
Lucas & Renzaglia (2002) concluded that hornwort
stomata open once through osmotic changes, thereafter remaining open and with a primary role
in providing a passageway for gaseous exchange
rather than regulating transpirational flow. This
conclusion fits well with the anatomy of hornwort sporophytes, characterized on the one hand
by the lack of an endohydric water-conducting
system (Ligrone et al., 2000) and on the other by
schizogenous formation of intercellular spaces
interior to stomata.
As part of an ongoing investigation of stomatal
function in hornworts, necessitating observations of
the dimensions of many thousands of these, we
recorded several novel features of guard cell development and the regular occurrence of abnormal
stomata associated with the dehiscence grooves along
the sporophytes. Our findings and their possible
implications for stomatal function and evolution in
hornworts are the basis for this paper.

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Materials and Methods


Specimen collection and preparation
Freshly collected mature sporophytes of Anthoceros
agrestis Paton, A. punctatus L., Phaeoceros carolinianus (Michx.) Prosk. (all from southern England), P.
laevis (L.) Prosk. (from Berkeley, California, USA)
and Megaceros flagellaris (Mitt.) Steph. (from Lake
Ianthe, South Island, New Zealand) were excised
from the subjacent thalli level with the bases of the
involucres and sliced longitudinally at right angles to
the dehiscence grooves. The two halves were mounted
in water, epidermis upwards, and digital images taken
of all the stomata, recording the location of the
groove when abnormal morphology was found, using
a Zeiss Axioscop 2 microscope equipped with an
AxioCam MRc digital camera. Starch was visualized
by Lugols iodine staining reagent (Iodine in potassium iodide solution) (Sigma). Sporophytes were
harvested and blanched in 80% (vol/vol) ethanol.
After rinsing with double-distilled water the sporophytes were stained with Lugols reagent and briefly
rinsed with water. Stained sporophytes were imaged
as described above.

Confocal microscopy
For confocal imaging, fresh samples of the four hornwort
species (sporophytes), the moss species Bryum radiculosum Brid. (sporophyte) and Polytrichastrum formosum
(Hedw.) G.L.Sm. (stem) and of Prunus (stem) were cut
into thin sections and placed onto a microscope slide in a
droplet of water and sealed with a coverslip and nail
varnish to prevent dehydration. Confocal images were
acquired with a Nikon A1-Si laser-scanning confocal
microscope using either a 10x objective (NA50.3) or
60x oil-immersion objective (NA51.4). Images were
recorded with pixel dimensions of 1.24 mm (for 10x
objective) and 0.21 mm (for 60x objective). To investigate the presence and possible distribution of lignin the
phloroglucinol/HCl histochemical test (which selectively
quenches lignin autofluorescence; Biggs, 1985) was used
in conjunction with confocal microscopy. Stems of the
moss P. formosum (known to contain lignin; Ligrone
et al., 2008) and of Prunus were used as controls. Lignin
fluorescence was excited with a 405-nm line of 100mW
cube laser (Coherent Inc., USA, http://www.coherent.com) and a 488-nm line of 50mW sapphire laser
(Coherent Inc., USA, http://www.coherent.com) was
used for exciting chlorophyll fluorescence. The fluorescence signal was collected with a 32-channel spectral
detector at 10nm resolution on a diffraction grating
covering a range of wavelengths between 410 nm
730 nm. Stomata were visualised using a 34.5 mm (1.2
airy units) confocal pinhole and a number of z-stacks
(typically between 60 and 130) with optical thickness of
125 nm each were acquired. The fluorescence signal on
collected raw images was spectrally unmixed with a
Nikon NIS-Elements software (http://www.nis-elements.

Pressel et al.

com/) on a basis of pure chlorophyll a (http://omlc.


ogi.edu/spectra/PhotochemCAD/index.html) and lignin (Albinsson et al., 1999) emission spectra which
were used as references. The unmixed fluorescence
signal from each z-stack was then projected onto a
maximum projection image.

Conventional scanning electron microscopy


(SEM)
For SEM, sporophytes were dehydrated in a 1 : 1
ethanol: acetone series, critical-point dried using CO2
as the transfusion fluid, sputter-coated with 390 nm
palladium-gold (AuPd) and viewed using a Philips
XL30 scanning electron microscope (Pressel &
Duckett, 2006).

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Cryo-scanning electron microscopy (cryo-SEM)


For cryoSEM, sporophytes were mounted on an
aluminium stub using Tissue-Tek (Sakura Finetek,
Zoeterwoude, Netherlands) and plunged in liquid
nitrogen slush to preserve their hydrated and partially
dehydrated states in a frozen condition in a Gatan Alto
2500 cryosystem (Abingdon, UK). The frozen sporophytes were vacuum-transferred to a high vacuum
cryogenic preparation chamber to prevent contamination and the build-up of ice. Ice was sublimed off the
surface by raising the temperature to 290uC for 5 min.
The samples were then cooled to 2130uC and AuPd
sputter coated with a cold magnetron sputter coater.
The coated sporophytes were then inserted directly into
the SEM via an airlock to avoid ice build-up and to
maintain their frozen state. Inside the SEM, the
samples rested on a cold stage with the temperature
maintained at 2130uC. A FEI Quanta 3D FEG dual
beam microscope with an Oxford Instruments energy
dispersive spectroscopy (EDX) attachment was used to
image the sample at 30kV and beam current of 0.11nA
(Duckett et al., 2009).

Observations
Light microscopy
The early ontogeny of the stomata in Phaeoceros is
illustrated in Figure 1. Nascent stomata mother cells,
only 13 mm above the base of the sporophytes, can
be identified by the presence of a single small
spherical to ovoid chloroplast, less than 5 mm in
diameter lying usually along a side wall (Figure 1a),
in contrast to early fragmentation of the chloroplasts
in all the other epidermal cells which occurs immediately above the foot. At this stage in sporophyte
ontogeny the epidermal cells have reached only O of
their final lengths, but are already the same width as
those in the mature sporophytes. The lengths of the
stomata mother cells are not appreciably different
from the other epidermal cells but these tend to be
slightly wider than the other epidermal cells. The
mother cell chloroplast then divides and initially the
two daughter chloroplasts, still less than 5 mm in

Stomatal differentiation in hornworts

diameter, lie side-by-side in various positions along


the cell walls (Figure 1 bd). Further elongation of
the mother cell to L its final size sees the spherical
chloroplasts increasing in size to diameters of around
10 mm and their migration to lie side-by-side in the
centre of the cells (Figure 1e). Longitudinal division
of mother cells, approximately half way up the
involucres, between the two chloroplasts (Figure 1f,
6b) coincides with the nascent guard cells and the
other epidermal cells reaching their final lengths.
Towards the top of the involucres the epidermal cells
increase slightly in width (13.1 +
2.3 mm) except for
those in the two dehiscence grooves which are
markedly narrower (8.3 +
1.4 mm). Guard cell
maturation sees these increase slightly in width to
around 15 mm at their mid-point.
The subsequent cytological changes occurring in the
stomatal guard cells are illustrated in Figure 2. The
previously spherical guard cell chloroplasts become
highly pleiomorphic and increase in size to often
exceed 20 mm in length (Figure 2a, b). In contrast to
the starch-free chloroplast fragments less than 2 mm in
diameter which are scattered in the peripheral
cytoplasm of the neighbouring cells, the guard cell
chloroplasts accumulate abundant small starch grains
as revealed by black staining with Lugols iodine
(Figure 4g). These massive chloroplasts are most
frequently positioned immediately below the outer
wall and most often in close proximity to the position
of the developing pores (Figures 2ac; 5a). Coinciding
with the opening of the stomatal apertures, the
chloroplasts, still with a highly pleomorphic outline,
divide (Figure 2d, e). They then lose the starch and
become spherical again (Figures 2f, g; 5b) with
diameters of 11.3 +
2.4 mm (P. laevis) just slightly
smaller than the two in each of the subepidermal cells
(13.9 +
2.0 mm, in P. laevis) and typical of the
sporophytes of Anthoceros and Phaeoceros.
The differences between the guard cell and other
epidermal and subepidermal chloroplasts in hornworts
are most clearly seen from red chlorophyll autofluorescence using confocal microscopy (Figure 5ad). The
same fragmentation of the chloroplasts of the sporophyte epidermal cells also occurs in the estomate genus
Megaceros (Figure 5c, d). In contrast to the hornworts, both the guard cells and adjacent epidermal
cells in mosses (Figure 5e) contain spherical or ovoid
chloroplasts which are slightly larger and more
numerous in the guard cells. The larger size of those
in the guard cells is due to their higher starch content
(Sack & Paolillo, 1983).
In all four hornwort species examined, the pores
open either immediately below the top of the
involucres or at the latest within 12 mm above it.
The nascent pores (width 12 mm) are short in length
(1015 mm) and have thin walls. The numbers of these

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Stomatal differentiation in hornworts

Figure 1 Early ontogeny of the stomata of Phaeoceros carolinianus. The apex of the sporophytes is beyond the top of each
image. Note the absence of an intact single chloroplast in all the epidermal cells other than those forming the stoma.
a) Stomatal mother cell containing a single spherical chloroplast on the lateral wall (arrowed); b-d) dividing chloroplasts
(arrowed) in various positions in stomatal mother cells; e) chloroplasts becoming aligned side-by-side (arrowed) immediately
prior to the division of the stomatal mother cell; f) nascent stoma with spherical centrally located chloroplasts (arrowed). Scale
bars: 50 mm.

newly opened stomata vary from between 23 to up to


810 per sporophyte. Subsequently the pores increase
in size and attain their maximum dimensions (e.g.
Phaeoceros leaevis: width 10 +
2 and length 37 +
5 mm;
Anthoceros puncatus: width 8.5 +
1.5
and
length
24.2 +

3 mm) usually less than 23 mm above the top of the


involucres. Further maturation sees the development
of wall thickenings bordering the pores, the accumulation of dense wall material in and above the apertures
and migration of the two chloroplasts to the inner walls
(Figures 2hj; 6gj). That profound changes in the cell
walls accompany stomatal maturation is further
demonstrated by our fluorescence data (Figure 5fi).

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In young unopened stomata it is only the walls lining


the nascent pore that exhibit a blue autofluorescence
component spectrally close to lignin (Figure 5f, g),
whilst in mature, open stomata, this blue component
becomes widespread over the outerwalls of the guard
cells (Figure 5h, i). However, the blue autofluorescence
component remains unchanged after treatment with
phloroglucinol (Figure 5j), whilst it is quenched
completely in the stems of both Polytrichastrum
formosum and Prunus (Figure 5kn)
The most remarkable features of our observations
were: 1) the extreme regularity of stomatal spacing
both along and between sporophytes; 2) the invariable

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Figure 2 Maturational changes in the stomata of Phaeoceros


laevis. ag) Stomata below or just below the rim of the
involucre. Note the absence of thickenings around the
apertures and chloroplasts in focus just below the outside
walls; ac) single pleiomorphic chloroplasts; d, e) dividing
chloroplasts; f, g) the chloroplasts become spherical following division; hj) fully differentiated stoma. Note the accumulation of dense material in the apertures and the out of focus
chloroplasts along the inner walls. Scale bars: 50 mm.

Stomatal differentiation in hornworts

occurrence of two guard cells; 3) the absence of


mature stomata with closed apertures and the precise
alignment of the guard cells parallel to the adjacent
epidermal cells. It should also be noted that all the
normal stomata illustrated in Figure 2 are highly
symmetrical and longitudinally-aligned with the wall
between the two guard cells most often meeting the
middle of the end walls of the adjacent cells at each
end. Shorter more rounded stomata with varied
dispositions are however frequent at the tips of
young undehisced sporophytes where epidermal cell
patterning is less precise (Figure 6h, i).
A further constant feature between sporophytes and
species is the pattern of spore maturation. Level with
the top of the involucres the developing spores are at
the young tetrad stage (Figure 8b) with maturation
taking place within the first centimetre above this
(Figure 8d). This level, where the spores mature,
remains the same irrespective of the length of the
sporophytes and whether or not they have dehisced.
A representative selection of abnormal stomata in
Phaeoceros laevis is illustrated in Figure 3. These show
that those with the groove to the right (Figure 3aj)
are mirror images of those with it to the left
(Figure 3kt). All but 18 of the 135 abnormal stomata
we observed conformed to this pattern of dextral and
sinistral asymmetry. In a few cases, only one end of the
dividing wall between the guard cells was displaced
either away from the grooves (Figure 3a and k) or
more often towards the grooves (Figure 3b and l).
Displaced walls at both ends were the commonest
abnormality (Figure 3ch and mr). In every case the
longer outer wall of the upwardly displaced guard cell
lies away from the groove. Even more pronounced
displacement of the dividing wall to nearly 45 degrees
from the long axis of the sporophytes produced pairs
of short fat guard cells (Figure 3i, j and s, t). The gross
structural abnormalities described above appear to
have no effect on the normal course of guard cell
differentiation. Thus we recorded the full range of
cytological changes from young stomata with single or
two pleiomorphic chloroplasts (Figure 3b, c, f, j, m, p,
q), to those with two spherical ones below the outer
walls (Figure 3d) to those with two chloroplasts lying
along the inner walls at maturity (Figure 3a, g, h, l, t).
Apertures also developed normally and accumulated
additional wall material at maturity (Figure 3a, b, i, t).
Exactly the same kinds of stomatal abnormality,
described above in Phaeoceros laevis, were associated
with the dehiscence furrows in P. carolinianus,
Anthoceros punctatus (both not illustrated) and A.
agrestis (Figure 4ae). Here as in P. laevis, displaced
walls in the guard cells on the left of the grooves
(Figure 4a, b) mirrored those on the right (Figure 4ce).
In over 50 sporophytes examined (nearly 3000
stomata) during this study we observed paired

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Figure 3 Phaeoceros laevis. Abnormal stomata lining the sporophyte grooves (G). The tip of the sporophytes is towards the
top of the page in every image. aj) Stomata on the left hand-side of grooves; (kt) stomata on the right hand-side of grooves.
These are mirror images of those on the left. Positions where abnormal walls separating the guard cells meet adjacent cells are
arrowed; a, k) wall between the guard cells meeting the cell above normally but with the lower end abutting the cells away from
the grooves; b, c and l, m) wall between the guard cells meeting the cell below normally but with the upper end abutting the cell
lying towards the grooves; d, n) stomata with displaced guard cells, the upper with its outer wall lying away from the groove;
e-h and o-r) sigmoid walls between guard cells: in every case the outer wall of the guard cell displaced upwards lies away from
the grooves; i, j and s, t) near 45 degree divisions have produced short fat guard cells. Scale bars: 50 mm.

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whilst those of mature ones exhibit a rough irregular


surface (Figure 6gi) with the additional wall material often completely covering the pores (Figure 6h,
i). Cryo-SEM reveals that the pores are lined with
wax rodlets (Figure 6j). These are removed by the
organic solvents used in critical point drying for
normal SEM but the irregular deposits of external
wall material are unaffected (Figure 6h, i).
Alongside the maturational changes that accompany stomatal development are equally profound
changes in the subtending sporophytic intercellular
spaces (ISs) (Figures 7 and 8). Inside the involucre
and at the level where the young stomata have yet to
develop pores, the ISs are completely filled with
liquid (probably mucilage) (Figure 7a). Spores are at
the pre-meiotic stage, as evidenced by the presence of
spore mother cells (Figure 7b). It is only after pore
opening (Figure 7c) when spores are at the young
tetrad stage (Figure 8a, b) that the liquid in the ISs
begins gradually to dry out (Figures 7df and
Figure 8a). By the time they near maturity at the
late tetrad stage (Figure 8d) most if not all the liquid
in the ISs has dried out and has been replaced by gas
(Figure 8c). Liquid remains around the spores at this
stage and does not disappear until after dehiscence.

Summary of stomatal ontogeny


The key events in hornwort stomatal ontogeny are
shown diagrammatically in Figures 9 and 10. These
figures include the changes in positions, sizes and
shapes of the chloroplasts in relation to guard cell
ontogeny, the development of wall thickenings lining
the apertures, the deposition of additional material
over the external walls and wax rodlets inside the pores.
Also illustrated is the formation of initially mucilagefilled intercellular spaces and the replacement of liquid
by gas after the stomata open. Remnants of the
mucilage remain along the cell junctions.

Discussion

Figure 4 ae) Anthoceros agrestis abnormal stomata. a, b)


Groove on right; ce) groove on left. fh) Phaeoceros laevis.
f) Two stomata with guard cells lying together; g, h) abundant
starch grains in the chloroplasts of young guard cells as
revealed by black staining with Lugols iodine (g); lack of
staining in chloroplasts from mature guard cells (h). Scale
bars: (g, h) 100 mm; (af) 50 mm.

stomata with shared guard cell walls on only three


occasions in Phaeoceros laevis (Figure 4f) and only
once in Anthoceros agrestis (not illustrated).

Electron microscopy
SEM clearly shows the dehiscence groove and the
stomata widely scattered over the sporophyte surface
(Figure 6a). The guard cells of young stomata have
completely smooth external walls (Figure 6b, c, f)

This study has revealed several novel and unique


features of hornwort stomata which set these apart
from those in other groups of land plants and thus
become pertinent to considerations of both their
function and evolution.

Developmental considerations
Development from epidermal cells, otherwise morphologically indistinguishable, apart for their chloroplasts, from their neighbours up until the division
producing the two guard cells, is very different from
all the various patterns of cell division leading to
stomatal formation in vascular plants (Evert, 2006).
Even in the simplest case of anomocytic stomata,
where the epidermal cells around the guard cells are
not distinguishable from their neighbours and subsidiary cells are absent, the stomatal mother cells are
much smaller than the other epidermal cells (Evert,

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Figure 5 Confocal images showing major differences between the chloroplasts of the guard cells and those of other epidermal
cells. All images were spectrally unmixed (as described in methods) into separate blue and red autofluorescence components that
match the pure fluorescence emission spectra of spruce lignin and chlorophyll a, respectively.) ae) chlorophyll autofluorescence
component shown in red; fi, k, m) lignin or spectrally close to lignin autofluorescence component shown in blue and overlaid with
the red chlorophyll autofluorescence component; j, l, n) lignin or spectrally close to lignin autofluorescence component (blue) after
treatment with phloroglucinol overlaid with the red chlorophyll autofluorescence component; a) large pleiomorphic plastids
immediately below the outer guard cell wall in a young stoma; b) plastids in epidermal cells after fragmentation, those in the
subepidermal cells and in the guard cells are rounded; c, d) epidermal plastids in the estomate Megaceros flagellaris (d) also
fragment, whilst (c) the subepidermal ones remain intact; e) plastids in the epidermal and guard cells of the moss Bryum
radiculosum; f, g) young, unopened stomata of Anthoceros punctatus (f) and Phaeoceros laevis (g) the blue autofluorescence
component is restricted to the lining of the nascent pores; h, i) mature stomata of the same two species - the blue autofluorescence
component becomes widespread in the guard cell walls; j) sporophyte of Anthoceros punctatus after treatment with phloroglucinol
the blue autofluorescence component remains unchanged; k, l) stems of Polytrichastrum formosum, untreated (k) and treated (l)
with phoroglucinol the blue autofluorescence component is quenched by the treatment; m, n) stems of Prunus, untreated (m) and
treated with phloroglucinol (n) the blue autofluorescence component is quenched by the treatment. Scale bars: (m, n) 300 mm; (al)
50 mm.
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Figure 6 Scanning electron micrographs, all cryo except g and h (critical point dried). Anthoceros agrestis (a, c, g, h);
Phaeoceros laevis (b, e, f); Phaeoceros carolinianus (d, i, j). a) Fractured sporophyte showing a dehiscence groove (arrowed)
and widely scattered stomata (arrowed); b, c) young unopened normal (b) and abnormal stomata (c); d, e) abnormal stomata;
f) newly opened stoma; note the smooth surface of the guard cells; gi) older stomata with irregular deposits of wall material
over the guard cells; j) a coating of wax rodlets lining an open pore. Scale bars: (a) 200 mm; (bi) 30 mm; (j) 5 mm.

2006). Stomatal mother cells indistinguishable from


their neighbours appear to occur in some mosses
(Paton, 1957; Paton & Pearce, 1957) but in many
others the adjacent cells have a radial arrangement that
characterizes actinocytic stomata in angiosperms
(Evert, 2006). The stomata that most closely match
those in hornworts, because of their low densities,
longitudinal alignment and guard cells derived from

epidermal cells indistinguishable from their neighbours,


occur on the leafless axes of Devonian fossils (Edwards
et al., 1996, 1998).
The present observations clearly point to determining roles for the chloroplasts in guard cell ontogeny, a
finding closely in line with Brown and Lemmons
(1985a) account of monoplastidic mitosis in hornworts
where future cytokinetic planes and microtubule

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Figure 7 Cryo-scanning electron micrographs. Anthoceros agrestis. a) Fractured sporophyte, lower portion of within
involucre: all the intercellular spaces are liquid-filled. Stomata are unopened (arrowed) and spore mother cells (arrowed) (b)
have yet to undergo meiotic division; c) young, open stoma; the subtending intercellular spaces are all filled with liquid (*). It is
only after stomatal opening that the liquid in the ISs begins gradually to dry out (df) (arrows in d and f indicate gas-filled
spaces). Scale bars: (a) 100 mm; (bf) 50 mm.

systems are predicated by plastid division and positioning. As in other mitotic cells in hornworts, migration of
the two daughter chloroplasts to a central position in
the stomatal mother cells determines the future plane of
division. Thus the central side-by-side location of the
two chloroplasts in the stomatal mother cells ensures
their longitudinal division and the distribution of a
single chloroplast to each guard cell. This key role for

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the chloroplasts in stomatal ontogeny, clearly absent in


the polyplastic stomatal cells of mosses, invites
comparison with Selaginella (Brown & Lemmon,
1985b) where the guard cells also inherit a single
chloroplast from the mother cells which then undergo a
series of migrations and divisions. However, unlike
hornworts the stomatal mother cells in Selaginella are
morphologically distinct from the other epidermal

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Stomatal differentiation in hornworts

Figure 8 Cryo-scanning electron micrographs. Anthoceros agrestis. a) Fractured sporophyte, just above involucre. Liquid
gradually drying out from ISs. Spores are at the young tetrad stage (enlarged and arrowed in (b)); c) fractured sporophyte, 1cm
above involucre: most of the ISs are now gas-filled, stomata have irregular deposits of wall material over the guard cells
(arrowed) and spores are almost mature (enlarged and arrowed in (d)). Scale bars: (a, c) 100 mm; (b, d) 50 mm.

cells, the young guard cells chloroplasts in Selaginella


never go through a highly pleiomrphic starch-filled
phase in their ontogeny and the chloroplasts in the
other epidermal cells retain their integrity unlike their
fragmentation in hornworts. Overall these differences
are more indicative of parallel evolution rather than
homology in stomatal ontogeny between the two
groups.
In contrast to the extremely uniform stomatal
morphology generally, the regular occurrence of abnormalities lining the grooves is all the more striking
and clearly requires a possible explanation. Differential
elongation of the epidermal cells in the grooves versus
those outside these can be ruled out since all these cells
have reached their full lengths when the guard cell
division takes place. The most likely explanation lies in
pressure/tension differences on the guard mother cells
lining the grooves, as a result of differential radial
expansion of the epidermal cells. We envisage that such

pressure would displace the chloroplasts away from


their central side-by-side location and, with plastiddetermined cytokinesis (Brown & Lemmon, 1985a),
this would induce the abnormal division planes. The
mirror images of the guard cells on opposite sides of the
grooves fit this explanation. Interestingly we are not
aware of any previous reports of matching sinistral and
dextral stomatal abnormalities like those found in
hornworts. Whereas in most cases in vascular plants
morphological abnormalities in stomata are associated with death of some of the cells and failures in
pore development (Dehne, 1961; Takahashi, 1962;
Inamdar & Patel, 1969; Inamdar et al., 1969; Gopal &
Shah, 1970; Inamdar et al., 1970; Inamdar & Patel,
1976), in hornworts, whatever the changes in the
morphology of the guard cells, pores develop normally.

Functional considerations
The presence of waxes lining the pore chambers
(Figure 6j), a feature not previously noted in hornworts,

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Figure 9 Schematic representation of stomatal ontogeny in hornworts; tangential views (top) and transverse sections (bottom).
a) Nascent mother cells with a single spherical chloroplast (Fig. 1a); b) division of the mother cell chloroplast (Fig. 1 bd); c)
immediately before the longitudinal division of the mother cell the chloroplasts increase in size and migrate to lie side-by-side
centrally (Fig. 1e); d) nascent stoma with each guard cell containing a single spherical chloroplast (Fig. 1f); the initial stage in
aperture formation (Fig. 2ac) coincides with division of the subepidermal chloroplasts and the almost complete disappearance
of the fragmented epidermal cell chloroplasts. Huge pleiomorphic, starch-filled chloroplasts in the guard cells. Fluorescent
additional wall material above the aperture (Fig. 5f, g). Mucilage-filled intercellular spaces (Fig. 7a, c); f) pore formation coinciding
with division of the guard cell chloroplasts (Fig. 2df) and spread of fluorescent material over the external surface (Fig. 5h, i).

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Figure 10 Schematic representation of stomatal ontogeny in hornworts; cont. from Fig. 9. a) Two spherical chloroplasts near
the surface of each of the guard cells (Fig. 2g). The mucilage in the intercellular spaces below the epidermis has dried down to
the cell corners but remains between the cells further inside the sporophytes (Fig. 7d, f); b) mature stoma with irregular
deposits of fluorescent wall material covering the external surface (Figs. 5h, i; 6h, i); wax rodlets lining the pore (Fig. 6j) and
chloroplasts lying along the inner walls of the guard cells (Fig. 2hj). All the intercellular spaces are now gas-filled with drieddown mucilage along the cell junctions (Fig. 8a, c).

most likely prevents water entering the sporophytic air


chambers thus paralleling the function of the hydrophobic cuticular ledges around the pores in complex
thalloid liverworts (Schonherr & Ziegler, 1975).
However, our current cryo-SEM observations (Pressel
& Duckett, unpublished data) on moss stomata and on
the air pores in complex thalloid liverworts reveal that
waxes are widespread here also. While these waxes are
removed by the organic solvents used in critical point
drying, the additional wall materials that accumulate
over the sporophyte surface and particularly the guard
cells in hornworts are not. Their recalcitrant nature and

the fact that they often completely cover the pore lumina
seriously calls into question the ability of hornwort
stomata to open and close and thus to respond to
external stimuli as reported by other authors (Hartung
et al., 1987, 1994; Bopp & Werner, 1993; Christianson,
2000). Our confocal data (Figure 5) further suggest
substantial developmental changes in the guard cells,
with blue fluorescing compounds associated exclusively
with the developing pores in young stomata but
becoming widespread in the outer walls of the guard
cells of mature ones. Our preliminary histochemical tests
indicate that these are not lignin-like compounds whose

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fluorescence is quenched upon phloroglucinol treatment


as initially hypothesised given previous reports of
lignin-like compounds in hornwort sporophytes
(Ligrone et al., 2008). Thus the nature of hornwort
guard cell walls and the changes that accompany
stomatal development remain to be determined.
However, irrespective of the nature of the changes in
the walls of the guard cells which accompany stomatal
development, our observations that the sporophytic
ISs are initially liquid-filled (Duckett et al., 2010) and
that liquid is gradually replaced by gas only after pore
opening, by which time the spores are at the tetrad
stage, give a totally new perspective to the possible
functioning of hornwort stomata. Whilst on the
strength of our structural and developmental data
alone we cannot exclude that young stomata do
respond to external stimuli, as reported before
(Hartung et al., 1987, 1994), any role of such movements in gaseous exchange would be impossible given
that, at this stage, the ISs are completely filled with
liquid. Thus, we can only conclude that pore formation, and indeed any possible movement, has a role in
sporophyte dehydration leading to dehiscence and
spore dispersal. Our observations that the sporophytic
intercellular spaces of hornworts are initially liquidfilled also clearly set hornwort stomata apart from
those in tracheophytes, where similar spaces in the
mesophyll are air-filled from the onset (Pressel &
Duckett, unpublished data). Our preliminary investigations on moss sporophytes are revealing that here
also the nascent ISs are liquid-filled (Duckett et al.,
2010; Pressel & Duckett, unpublished data).
The fact that we never observed closed mature
stomata and found a rather narrow range of mature
aperture dimensions reinforces the earlier conclusion by
Lucas & Renzaglia (2002) that hornwort stomata open
once and never close. Recent comparative cryo-SEM
studies have shown that whereas stomatal apertures in
angiosperms are greatly influenced by epidermal turgor,
those of lycophytes, ferns and conifers are not affected
(Franks & Farquhar, 2007; Brodribb & McAdam, 2011).
The fact that hornwort stomata invariably remain open
post dehiscence when the epidermal cells are highly
desiccated and as the older parts of the sporophytes are
drying out indicates that these are like those in
pteridophytes and conifers. In addition, in hornworts
mechanical constraints against aperture changes
imposed by both the additional wall materials over the
guard cells and the thickened longitudinal radial walls of
the adjacent epidermal cells would appear to be
considerable. Our parallel experiments to those of
Franks & Farquhar (2007) on the effects of drought on
young hornwort stomata (Pressel & Duckett, unpublished data) show these to be equally non-responsive.
The prominence of the guard cell chloroplasts early
in guard cell ontogeny is most likely related to the

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synthesis of both the organic osmoticum that generates opening (Lucas & Renzaglia, 2002) and to the
synthesis of the additional wall materials rather than
providing the energy source for active opening and
closing via a potassium pump. Whilst the present study
confirms Cavers (1911) original observation that the
mature guard cells contain 2 chloroplasts typical of
sporophytic cells, the fragmentation of the other
epidermal chloroplasts in Anthoceros, Phaeoceros
and Megaceros, which lacks stomata, appears to be
unique to these cells in the hornwort life cycle (Duckett
& Renzaglia, 1988).
The contrast between the ontogeny of the guard
cell and epidermal cell chloroplasts in hornworts
from that in mosses is perhaps suggestive of possible
functional differences between the two groups. The
presence of similar chloroplasts throughout the
epidermis of moss sporophytes (Sack & Paolillo,
1983) may perhaps relate to active opening and
closing recently reported by Chater et al. (2011) in
Physcomitrella and Funaria in response to CO2
concentrations, ABA, darkness and fusicoccin (an
activator of stomatal opening) and using mutants in
the former, in contrast to earlier experimental studies
that showed either ambiguous (Garner & Paolillo,
1973) or no responses (Paton & Pearce, 1957)
though here the negative results are now attributed to
inappropriate techniques. From their results Chater
et al. (2011) argue that core regulatory components
involved in guard cell signalling are common to both
angiosperms and mosses and originated in their
common ancestor over 400MYA. Presenting similar
data on Selaginella, Ruszala et al. (2011) come to the
same conclusion. In striking contrast Brodribb and
McAdam (Brodribb & McAdam, 2011; McAdam &
Brodribb, 2012a,b) found that lycopods and ferns
lack the key responses to ABA and epidermal cell
turgor and present the counter argument that active
control of water balance by stomata evolved after the
divergence of the ferns 360MYA. Our developmental
data showing that hornwort stomata never close, not
to mention the very different origin of the intercellular spaces from those in vascular plants, are
more closely in line with the views of Brodribb and
McAdam and are further underlined by our ongoing
experiments where we have failed to detect significant
stomatal responses in hornworts to ABA, desiccation
and darkness, not to mention the absence, as in
Sphagnum (Duckett et al., 2009), of potassium fluxes
between the guard cells and adjacent epidermal cells
(Pressel & Duckett, unpublished data). To add fuel to
this ongoing and unresolved debate it should be
noted that most of experimental treatments in Chater
et al. (2011) induced only small changes in stomatal
aperture areas (which translate into only very slight
changes in aperture widths) and none induced

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complete closure. Possible active control in mosses


also needs to be explained in relation to initially
liquid-filled intercellular spaces and whether or not
opening and closing involves potassium pumps.
The close developmental relationship between
stomatal ontogeny and sporophyte maturation described here in hornworts also points to a possible
functional relationship. The rapid maturation and
opening of the stomata above the involucres provides
direct access to atmospheric carbon dioxide but only
after the liquid in the ISs has dried out. Just how far
atmospheric carbon dioxide, fixed by the sporophytes, contributes directly to the developing spores
is also called into question by the radiotracer study
by Thomas et al. (1978). Whilst carbon fixed by the
sporophytes is sufficient for self-maintenance, continued growth depends on nutrient transfer from the
parent gametophytes across the placenta (Ligrone
et al., 1993). In fact, the greatest percentage of
radioactivity, derived from labelled glucose fed to the
gametophytes, is in the spores (Thomas et al., 1978).
These data, taken together with the fact that when
stomata first open, the ISs are filled with liquid and
the spores are already at the tetrad stage, and by the
time this liquid has been replaced completely by gas
spores are at the late tetrad stage, indicate that the
bulk of their biomass is derived from placental
nutrition rather than sporophyte photosynthesis. It
is also noteworthy that in the hornwort genera
Nothoceros, Megaceros, Dendroceros and Notothylas
that lack stomata, spore maturation usually occurs
when the sporophytes are deep within the involucres
(Schuster, 1984).
These nutritional/developmental considerations,
taken together with our structural data pointing to
fixed apertures, suggest that the principal role of
stomata in hornworts is one other than photosynthetic gaseous exchange. Perhaps the more plausible
explanation is that the stomata in hornworts facilitate
sporophyte desiccation, as is the case in Sphagnum
but here, unlike in hornworts (Duckett et al., 2009)
the pseudostomata lack localized wall thickenings
and pores that open (Boudier, 1988) and are not
subtended by intercellular spaces. In hornworts this is
all the more likely since they lack any kind of waterconducting cells that might maintain transpirational
water flow. From a very recent study of stomatal
development, in two highly contrasting moss sporophytes (Oedipodium and Ephemerum), Merced and
Renzaglia (2013) conclude that capsule anatomy
coupled with the exclusive existence of stomata on
capsules supports the concept that stomata in mosses
may also facilitate drying and spore dispersal.
One final remarkable feature of hornwort stomata
as revealed by the present study and the numbers
given for Folioceros by Asthana and Srivastava (1991)

Stomatal differentiation in hornworts

are their low densities compared to figures from extant


vascular plants. Thus in the present study, where we
recorded every stoma in the first 10 mm of the
sporophytes above the involucres, the numbers in all
four species varied between 1.52.66.3/mm2 and
numbers in Folioceros range from 10 in F. assamicus
to around 24 in other species, the typical range in
vascular plants is 1001000/mm2 (Evert, 2006). Two
possible explanations come to mind: 1) the low
densities reflect the ancient origins of hornworts over
400MYA (Villarreal & Renner, 2012; Magallon et al.,
2013) when the atmosphere contained much higher
carbon dioxide levels than at the present day (Beerling,
2007). Stomatal densities are likewise very low on the
leafless axes of palaeozoic fossils (Edwards et al., 1996,
1998); 2) the low densities reflect the possible primary
role of hornwort stomata in sporophyte desiccation
leading to dehiscence and spore discharge rather than
regulation of gaseous exchange. This being the case
any relationship between ambient carbon dioxide
levels and density would seem unlikely. The wide
range of stomatal numbers found in mosses, even
between related species growing in similar habitats
(e.g. in the Polytrichales: from absent in Pogonatum
and Atrichum to numbers ranging from approximately
20 in Polytrichum strictum to nearly 200 in
Polytrichastrum formosum (Paton & Pearce, 1957),
also calls into question a primary role in gaseous
exchange despite reports of physiological responses
like those in vascular plants (Chater et al., 2011;
Ruszala et al., 2011). Indeed in Funaria the proportion
of permanently open stomata increases as the capsules
mature thus contributing to capsule desiccation prior
to spore discharge (Garner & Paolillo, 1973).

Evolutionary considerations
In a very recent review of functional relationships
between the two generations in mosses Haig (2013)
further discusses the possibility that the original
function of stomata might have been sporophyte
desiccation in preparation for spore dispersal prior to
their becoming regulators of gaseous exchange.
Concentrations of stomata around the bases of the
sporangia in several early tracheophytes (Edwards
et al., 1996) are in line with this reasoning. Haig sets
out different competing schemes for the evolutionary
origin of stomata: 1) a single origin in a common
ancestor of all bryophytes and tracheophytes as
previously proposed by Raven (2002) and Ligrone
et al. (2012). This scheme is supported by the
supposed shared mechanisms of stomatal control
between mosses and tracheophytes (Chater et al.,
2011; Ruszala et al., 2011) but presents a problem
of explaining secondary losses in the basal moss
clades Sphagnum, Takakia and Andreaea; 2) stomata
evolved twice, once in the ancestor of peristomate

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mosses and once in the common ancestor of


hornworts and their sister group the tracheophytes;
3) stomata of hornworts and tracheophytes are not
homologous (Pressel et al., 2011) implying at least
three origins of stomata, a scenario closely on a par
with the multiple origins of water-conducting cells in
mosses, liverworts and tracheophytes (Ligrone et al.,
2000). The present study favours the third hypothesis.
Further resolution of these open questions about
stomatal development, function and evolution are now
being explored in mosses. The first phylogenomic
studies of stomatal genes, that include Physcomitrella,
identify two copies of FAMA, a gene involved in guard
cell specification and differentiation in Arabidopsis
(Bergmann & Sack, 2007; Peterson et al., 2010).
Similarly, transcriptomic analyses of the sporophytes
of Physcomitrella suggest a deeply conserved mechanism of stomatal development and control (Rychel &
Peterson, 2010; ODonaghue et al., 2013; Rudall et al.,
2013). Consequently, understanding the genetic control
of guard cell differentiation, spacing and pore formation is close at hand, at least for mosses. Looking to the
future in the post genomic era, further transcriptomic,
proteonomic and developmental approaches, together
with the availability of a hornwort genome in the near
future, should tell us whether stomatal biology has the
same genetic basis across land plants.
It will also be of interest to explore the effects of
different carbon dioxide concentrations on stomatal
density in hornworts. The only experiments to date on
the effects of elevated carbon dioxide concentrations in
bryophytes are on the moss Leptobryum pyriforme
(Baars & Edwards, 2008). These gave somewhat
equivocal results: whilst stomatal density and size
decreased under elevated carbon dioxide their number
per capsule was unchanged. These differences are
probably best interpreted as general growth responses
with stomata formation and differentiation preprogrammed within the closed determinate development of moss sporophytes. The results of similar
experiments on hornworts where stomata are produced continuously may be very different.

Acknowledgements
Zophia Ludlinska (Nanovision Centre, Queen Mary
University of London) for skilled technical assistance
using the cryo-SEM. Travel funds to SP from the
Natural History Museum and a Leverhulme Emeritus
Fellowship to JGD enabled the collection of the
materials used in this study.
Taxonomic Additions and Changes: Nil.

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