The evolution of the stomatal apparatus

Cryo-scanning electron micrographs of freeze-fractured hornwort gametophytes (a–c) and sporophytes (d–i): Anthoceros agrestis(a,c,d–f); Folioceros fusiformis (b); Leiosporoceros dussii (g); Megaceros enigmaticus (h); Dendroceros granulatus (i). Sections through thalli showing mucilage-filled cavities (asterisk). (cNostoccolony. (d,g) Intercellular spaces are initially liquid-filled (asterisk) but become gas-filled (e, arrowed) following stomatal opening. (f) Columella with gas-filled (asterisk) intercellular spaces. (h,i) Young (h) and mature (i) sporophytes of astomate taxa, showing complete absence of intercellular spaces in the assimilatory layers which collapse and dry (i). Scale bars: (a,b) 200 µm; (d,e,g) 50 µm; (c,f,h,i) 20 µm.

The evolution of the stomatal apparatus: intercellular spaces and sporophyte water relations in bryophytes—two ignored dimensions

by Duckett J. G., Pressel S. (2017)

Jeffrey G. Duckett, Silvia Pressel,


In Phil. Transact. Roy. Soc. B. 2017 –

Cryo-scanning electron micrographs of freeze-fractured moss sporophytes: Physcomitrella patens (a,b); Physcomitrium pyriforme(c,d); Lyellia crispa (e,f). (a,c) Young sporophytes with liquid-filled (asterisk) intercellular spaces. (e) Gas (arrowed) gradually replaces their initially liquid-filled content following stomatal opening, as evidenced by the presence of intercellular spaces only partially filled with liquid (asterisk in f). Liquid is first lost from the substomatal cavities (b; S, stoma) until the entire intercellular space system becomes gas-filled (d). Scale bars: (c,d) 100 µm; (a,e) 50 µm; (b,d) 20 µm.


Cryo-scanning electron microscopy shows that nascent intercellular spaces (ICSs) in bryophytes are liquid-filled, whereas these are gas-filled from the outset in tracheophytes except in the gametophytes of Lycopodiales. ICSs are absent in moss gametophytes and remain liquid-filled in hornwort gametophytes and in both generations in liverworts. Liquid is replaced by gas following stomatal opening in hornworts and is ubiquitous in moss sporophytes even in astomate taxa.

Cryo-scanning electron micrographs of freeze-fractured moss sporophytes: Polytrichum juniperinum (a,b); Mnium hornum (c); Atrichum undulatum (d); Pogonatum aloides (e,f). (a,b) Unopened (a) and open (b) stoma subtended by a gas-filled intercellular space. (c) Sunken stoma subtended by a liquid-filled intercellular space. (d–f) In astomate taxa, intercellular spaces are also initially liquid-filled (asterisk, e) and the same process of liquid replacement by gas occurs in their fully expanded capsules (d,f). Scale bars: (f) 200 µm; (a–e) 20 µm.

New data on moss water relations and sporophyte weights indicate that the latter are homiohydric while X-ray microanalysis reveals an absence of potassium pumps in the stomatal apparatus. The distribution of ICSs in bryophytes is strongly indicative of very ancient multiple origins. Inherent in this scenario is either the dual or triple evolution of stomata. The absence, in mosses, of any relationship between increases in sporophyte biomass and stomata numbers and absences, suggests that CO2 entry through the stomata, possible only after fluid replacement by gas in the ICSs, makes but a minor contribution to sporophyte nutrition. Save for a single claim of active regulation of aperture dimensions in mosses, all other functional and structural data point to the sporophyte desiccation, leading to spore discharge, as the primeval role of the stomatal apparatus.

Stomata in bryophytes

The function and evolution of stomata in bryophytes

by Duckett J. G., P’ng K. M. Y., Renzaglia K. S., Pressel S. (2010)

Jeff Duckett, Ken P’ng, Karen Renzaglia & Silvia Pressel

Jeff Duckett & Silvia Pressel, School of Biological
and Chemical Sciences, Queen Mary University
of London E1 4NS, UK;

Ken P’ng, Department of
Materials, Queen Mary University of London

Karen Renzaglia, Plant Biology Department, Southern
Illinois University, Carbondale, IL 62901, USA

v Fig. 1. Cryo-SEM images,
surface views. (a, b)
Paraphymatoceros minutus.
(a) Closed stoma from inside
an involucre; (b) newly opened
stoma immediately above
an involucre. (c) Phaeoceros
laevis, open stoma flanked
by desiccated and shrunken
epidermal cells well above
the dehiscence point on a
sporophyte. Bars, 20 μm.

v Fig. 2. Cryo-SEM images,
cryo-fractured preparations.
(a, b) Anthoceros agrestis.
(a) Sporophyte within an
involucre with mucilage-filled
intercellular spaces (arrowed)
in the assimilatory tissues; (b)
gas-filled intercellular spaces
(arrowed) above an involucre;
spores and pseudoelaters
embedded in mucilage (S). (c)
Podocarpus nivalis, gas-filled
intercellular spaces in a very
young leaf before stomatal
ontogeny. Bars, (a, c) 20 μm,
(b) 50 μm.

In Field Bryology,, 101: 38–40 –

It was most fitting that this celebration of Jean
Paton’s 80th birthday should include a presentation
on stomata. Two of Jean’s most seminal early works
were on stomata in British bryophytes (Paton, 1957;
Paton & Pearce, 1957); 52 years later these remain
indispensably authoritative accounts. Apart from a
flurry of excellent cytological and functional studies
– Sack & Paolillo (1973) and Garner & Paolillo (1973)
showed that the stomata of Funaria are able to open
and close, at least when young and that, like those
in higher plants, they are sensitive to abscissic acid;
while Maier’s (1974) and Boudier’s (1988) findings
that the stomata of Sphagnum lack subsomatal
air spaces lead to the conclusion that they should
best be considered as pseudostomata (Cox et
al., 2004) – the last half century saw virtually no
new advances in our understanding of bryophyte
stomata. This was almost certainly due to a lack
of suitable experimental technologies; thus, Jean’s
decision to leave stomata and thereafter pursue
liverworts was a very wise move.
It is widely assumed that stomata, one of the key
innovations in land plants, are homologous across
land plants (Raven, 2002). This hypothesis fits
neatly with 21st century total evidence phylogenies
that place liverworts firmly at the base of the land
plant tree (Shaw & Renzaglia, 2004; Renzaglia et
al., 2007). Accordingly, stomata first evolved in
mosses (± Sphagnum) and are thereafter present
from polytrichalean mosses and hornworts into
higher plants. Embedded in this notion of stomatal
homology is a further assumption that their principal
function (and the selection pressure driving the
same) is regulation of gaseous exchange (Raven,
2002), though this is certainly not the case in
Sphagnum where the pseudostomata are covered
with the calyptra until the sporophytes are almost
mature (Cox et al., 2004; Duckett & Ligrone, 2004;
Duckett et al., 2009).


Stomata are present in numerous lineages of moss with varying amino acid content and structures

Stomata in Bryophytes

by Randall J. M., McAdam S. (2019)

Randall Joshua M. [1], McAdam Scott [2].

1 – Purdue University, Botany and Plant Pathology, 915 W State St, West Lafayette, Indiana, 47907, United States
2 – Purdue University, 915 W State St, West Lafayette, Indiana, 47907, United States


In Botany 2019 –


Bryophytes, including mosses, are the oldest living group of land plants; therefore, they can be used to understand the origins of the organs that allowed the colonization of land.

Stomata are small openings found on the leaves of vascular plants to allow for water and carbon dioxide transfer, but in bryophytes the sexual organ, or sporophyte, has been found to also have stomata.

Using collected mosses from central Indiana, this study is intended to determine the presence of stomata across different moss lineages and their anatomy.

Mosses were keyed out using a dichotomous key according to gametophyte characteristics, and stomata were examined using a compound microscope to determine size and shape. Afterwards, a simple parsimony tree was created using this information to determine when stomata likely evolved.

Additional genomic information was collected from the 1000 Plant Project and BLASTPed against the SPEECHLESS (SPCH) transcription factor in Arabidopsis thaliana to find species across all lineages with similar proteins.

The SPCH transcription factor has been confirmed to allow for the development of guard cells that form stomata, and its presence in various moss lineages was used to build another phylogenetic tree.

Together, the physiological information and genomic analysis support the theory that modern stomata originated in non-vascular plants, but the history appears to be more complicated than previously thought.

Stomata are present in numerous lineages of moss with varying amino acid content and structures. The hypothesis of multiple losses and gains across mosses was not disproven.

Stomata in mosses do not require meristemoids, instead stomata differentiate before the capsule begins to expand

Fig. 1. Diagram of a stoma with an open and close pore. Stomata distribution at the base of the capsule of the moss Funaria.

Early land plants evolved a simple but effective mechanism to place stomata away from each other

by Merced A. (2016)


In Atlas of Science Nov. 20,2016 –

Fig. 2. Capsules of the moss Funaria. Colored scanning electron microscopy image of the tissue inside of the capsule that forms a labyrinth of air spaces.


Stomata are one of the key evolutionary features responsible for the successful colonization of land by plants.

A stoma is a pore surrounded by a pair of guard cells, when these cells are turgid and inflated the pore opens and when cells deflate the pore is closed.

This simple mechanism allows plants to optimize the amount of CO2 acquired for photosynthesis while reducing water loss due to transpiration.

Spacing of stomata in the epidermis, the external protective tissue of the plant, is important to ensure proper function and carbon fixation efficiency.

Mosses are of one of the firsts groups of plants to evolve stomata. Different from most plants, stomata of mosses and other early land plants are not located in leaves but on the spore producing capsule.

Our study investigated if the patterning and distribution of stomata in capsules of the moss Funaria follows a similar mechanism to that of flowering plants.

In flowering plants, namely the model organism Arabidopsis, stomata are place away from each other by a series of cell divisions of meristemoids, cells that actively divide to produce more meristemoids, epidermal cells or guard cells. This developmental process is regulated by genes and influence by the environment.

This study shows that stomata in mosses do not require meristemoids, instead stomata differentiate before the capsule begins to expand. Cells that will become stomata are align in files and separated by at least one cell. After the fate of the future stomata is decided, the surrounding cells divide perpendicular to it and differentiate into epidermal cells. Having stomata differentiate first, ensures that around 96-99% of stomata do not touch each other.

Stomatal pore and cuticle formation in Funaria 

Stomatal pore and cuticle formation in Funaria

by Sack F. D., Paolillo D. J. Jr (1983)

Boyce Thompson Institute for Plant Research and the Section of Plant BiologyCornell UniversityIthaca


In Protoplasma 116: 1–13 –


Cuticle and pore development in the guard cells of Funaria were investigated with the electron microscope.

Pore cuticle formation is simultaneous with the creation of the pore itself. The morphology of the pore cuticle is unlike that of any cuticle described in the literature. It has many lamellae which are penetrated by electron dense fibrils.

Three different cuticular morphologies exist from the pore to the subsidiary cell walls. The cuticles on the pore and outer walls contain fibrils that sometimes reach to the surface.

The subsidiary cell cuticle lacks fibrils altogether. It is hypothesized that

(1) cuticularization of the middle lamella contributes to ventral wall separation and

(2) differences in extent of cuticular fibrils are related to greater water loss from stomata than from subsidiary cells (peristomatal transpiration).

Abnormal stomata and undivided guard cell mother cells in Bryophyta



The effect of the calyptra on the plane of guard cell mother cell division in Funaria and Physcomitrium capsules

by French J. C., Paolillo D. J., Jr. (1975)


in Ann. Bot. 39: 233–236 – –


The calyptra influences the plane of division in guard cell mother cells of Funaria and Physcomitrium. Normally, capsules expand while sheathed by the calyptra and the axes of the stomata are parallel to the axis of the capsule in both genera.

Removal of the calyptra from an elongating sporophyte leads to seta thickening prior to capsule expansion and an essentially random orientation of stomata.

If the calyptra is removed from a sporophyte of Funaria at the time the division of the guard cell mother cells is expected, guard cells of abnormal shape and undivided guard cell mother cells are found in unusually high frequency.

Stomata in moss sporophytes

Screen Shot 2018-03-06 at 13.22.13
Phylogeny of major groups of mosses with the presence of stomata indicated by open circles. Taxa in which the sporophyte is enclosed within the epigonium until after meiosis are underlined. (A) Hypothesis in which there is a single origin of stomata from which pseudostomata of Sphagnum were derived. (B) Hypothesis in which stomata evolved twice and in which pseudostomata are not homologous to stomata.


Filial mistletoes: the functional morphology of moss sporophytes

by Haig D. (2013)

Department of Organismic and Evolutionary Biology, Harvard University, 26 Oxford Street, Cambridge, MA 02138, USA


in Ann Bot. 111(3): 337–345 – doi:  10.1093/aob/mcs295 –



A moss sporophyte inherits a haploid set of genes from the maternal gametophyte to which it is attached and another haploid set of genes from a paternal gametophyte. Evolutionary conflict is expected between genes of maternal and paternal origin that will be expressed as adaptations of sporophytes to extract additional resources from maternal gametophytes and adaptations of maternal gametophytes to restrain sporophytic demands.



The seta and stomata of peristomate mosses are interpreted as sporophytic devices for increasing nutrient transfer. The seta connects the foot, where nutrients are absorbed, to the developing capsule, where nutrients are needed for sporogenesis. Its elongation lifts stomata of the apophysis above the boundary layer, into the zone of turbulent air, thereby increasing the transpirational pull that draws nutrients across the haustorial foot. The calyptra is interpreted as a gametophytic device to reduce sporophytic demands. The calyptra fits tightly over the intercalary meristem of the sporophytic apex and prevents lateral expansion of the meristem. While intact, the calyptra delays the onset of transpiration.



Nutrient transfer across the foot, stomatal number and stomatal aperture are predicted to be particular arenas of conflict between sporophytes and maternal gametophytes, and between maternal and paternal genomes of sporophytes.