The evolution of the stomatal apparatus

Figure 3.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). (cNostoc colony. (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 Philosoph. Transactions Royal Soc. B Biol. Sci.

Figure 4.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.

Figure 5.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.

This article is part of a discussion meeting issue ‘The Rhynie cherts: our earliest terrestrial ecosystem revisited’.


The capsule dehiscence mechanism and the function of pseudostomata in Sphagnum

Exploding a myth: the capsule dehiscence mechanism and the function of pseudostomata in Sphagnum

by Duckett J. G., Pressel S., P’ng K. M. Y., Renzaglia K. S. (2009)

Jeffrey G. Duckett, Silvia Pressel, Ken M. Y. P’ng, Karen S. Renzaglia,

In New Phytologist 183: 1053– 1063 –


• The nineteenth century air‐gun explanation for explosive spore discharge in Sphagnum has never been tested experimentally. Similarly, the function of the numerous stomata ubiquitous in the capsule walls has never been investigated.

• Both intact and pricked Sphagnum capsules, that were allowed to dry out, all dehisced over an 8–12 h period during which time the stomatal guard cells gradually collapsed and their potassium content, measured by X‐ray microanalysis in a cryoscanning electron microscope, gradually increased. By contrast, guard cell potassium fell in water‐stressed Arabidopsis.

• The pricking experiments demonstrate that the air‐gun notion for explosive spore discharge in Sphagnum is inaccurate; differential shrinkage of the capsule walls causes popping off the rigid operculum. The absence of evidence for a potassium‐regulating mechanism in the stomatal guard cells and their gradual collapse before spore discharge indicates that their sole role is facilitation of sporophyte desiccation that ultimately leads to capsule dehiscence.

• Our novel functional data on Sphagnum, when considered in relation to bryophyte phylogeny, suggest the possibility that stomata first appeared in land plants as structures that facilitated sporophyte drying out before spore discharge and only subsequently acquired their role in the regulation of gaseous exchange.

A lack of physiological regulation of stomatal movements in hornworts compared with tracheophytes

Introduction to the Anthocerotophyta (UCMP), Berkeley Above, you can see pictures of the hornwort Phaeoceros. On the left is a plant with young sporophytes beginning to elongate from the top of the gametophyte. (



Hornwort stomata do not respond actively to exogenous and environmental cues

by Pressel S., Renzaglia K. S., Clymo R. S., Duckett, J. G. (2018) 

Silvia Pressel, Karen S. Renzaglia, Richard S. (Dicky) Clymo, Jeffrey G. Duckett


in Annals of Botany, mcy045 –


Backgrounds and Aims

Because stomata in bryophytes occur on sporangia, they are subject to different developmental and evolutionary constraints from those on leaves of tracheophytes. No conclusive experimental evidence exists on the responses of hornwort stomata to exogenous stimulation.



Responses of hornwort stomata to abscisic acid (ABA), desiccation, darkness and plasmolysis were compared with those in tracheophyte leaves. Potassium ion concentrations in the guard cells and adjacent cells were analysed by X-ray microanalysis, and the ontogeny of the sporophytic intercellular spaces was compared with those of tracheophytes by cryo-scanning electron microscopy.


Key Results

The apertures in hornwort stomata open early in development and thereafter remain open. In hornworts, the experimental treatments, based on measurements of >9000 stomata, produced only a slight reduction in aperture dimensions after desiccation and plasmolysis, and no changes following ABA treatments and darkness. In tracheophytes, all these treatments resulted in complete stomatal closure. Potassium concentrations are similar in hornwort guard cells and epidermal cells under all treatments at all times. The small changes in hornwort stomatal dimensions in response to desiccation and plasmolysis are probably mechanical and/or stress responses of all the epidermal and spongy chlorophyllose cells, affecting the guard cells. In contrast to their nascent gas-filled counterparts across tracheophytes, sporophytic intercellular spaces in hornworts are initially liquid filled.



Our experiments demonstrate a lack of physiological regulation of opening and closing of stomata in hornworts compared with tracheophytes, and support accumulating developmental and structural evidence that stomata in hornworts are primarily involved in sporophyte desiccation and spore discharge rather than the regulation of photosynthesis-related gaseous exchange. Our results run counter to the notion of the early acquisition of active control of stomatal movements in bryophytes as proposed from previous experiments on mosses.


Hornwort stomata are not actively regulated

Stomata, pores in the plant epidermis that regulate gas exchange, are a key innovation that enabled freshwater algae to colonize Earth’s landmasses some 500 Mya. Because stomata in bryophytes occur on sporangia, they are subject to different developmental and evolutionary constraints from those on leaves of tracheophytes. No conclusive experimental evidence exists on the responses of hornwort stomata to exogenous stimulation.

Hornwort stomata

Pressel and colleagues investigate stomatal behaviour in hornworts. They investigated responses of hornwort stomata to abscisic acid (ABA), desiccation, darkness and plasmolysis and compared these with those in tracheophyte leaves. Potassium ion concentrations in the guard cells and adjacent cells were analysed by X-ray microanalysis, and the ontogeny of the sporophytic intercellular spaces was compared with those of tracheophytes by cryo-scanning electron microscopy.

They show that there are no potassium fluxes associated with hornwort stomata, and that these do not respond to external factors (abscisic acid, desiccation, darkness and plasmolysis), which cause stomatal closure in other land plants. Their results run counter to the notion that active stomatal control was acquired early in the evolution of land plants, and support the alternative hypothesis of gradual acquisition of active control mechanisms.

Stomata in non-vascular land plants and CO2


Single-phylogram scenario illustrating key land plant lineages (bold text) and moss genera and the appearance of stomata in modern plants. Dashed lines indicate absence of stomata and solid black lines their presence. This phylogram with liverworts basal (Qiu et al., 20062007Liu et al., 2014) indicates a single origin of stomata and multiple losses, whereas an alternative topology with hornworts basal implies multiple origins (Haig, 2013Wickett et al., 2014).

Stomatal density and aperture in non-vascular land plants are non-responsive to above-ambient atmospheric CO2 concentrations

by Field K.J., Duckett J.G., Cameron D.D., Pressel S. (2015)

Katie J. Field, Jeffrey G. Duckett, Duncan D. Cameron, Silvia Pressel,

in Annals of Botany, 115 (6). 915 – 922. – –

Screen Shot 2017-11-23 at 16.39.50

Light (D, F, G, I–Q) and cryoscanning electron (A–C, H, R) micrographs of moss and hornwort stomata. (A, B) Physcomitrella patens: 12–14 stomata slightly irregularly spaced (e.g. the paired stomata in B) and randomly orientated around the capsule base; pores are round and subsidiary cells absent. (C, D) In the closely related F. hygrometrica the numerous stomata are axially orientated and regularly spaced. Also note the radial arrangement of the epidermal cells around the long-pored stomata (D); compare with hornworts (R). (E–G) Mnium hornum stomata sunk in deep pits. Note the liquid-filled subtending intercellular spaces (*) in (E). Stomata are often irregularly spaced [see the paired stomata in (F)] and have small round pores (F, G). (H–Q) Polytrichum juniperinum (H–K, grown at 440 p.p.m. [CO2]; L–Q, grown at 1500 p.p.m. [CO2]). Note the predominately axially arranged long-pored stomata frequently occurring in multiple groups (H–K). Abnormalities occur on almost all sporophytes and these increase under elevated CO2, as does the size of some of the apertures (L–Q). (J) A pair of stomata with a shared pore. (M–P) Stomata with abnormal pores. (O) Stoma with massive aperture. (P) Stoma with four guard cells. (R) Sporophyte of the hornwort A. punctatus. Note the regularly spaced axial stomata lacking subsidiary cells. Scale bars: (C, H, R) = 200 µm; (A) = 100 µm; (D–G, I–Q) = 50 µm; (B) = 20 µm.



Following the consensus view for unitary origin and conserved function of stomata across over 400 million years of land plant evolution, stomatal abundance has been widely used to reconstruct palaeo-atmospheric environments. However, the responsiveness of stomata in mosses and hornworts, the most basal stomate lineages of extant land plants, has received relatively little attention.

This study aimed to redress this imbalance and provide the first direct evidence of bryophyte stomatal responsiveness to atmospheric CO2.


A selection of hornwort (Anthoceros punctatus, Phaeoceros laevis) and moss (Polytrichum juniperinum, Mnium hornum, Funaria hygrometrica) sporophytes with contrasting stomatal morphologies were grown under different atmospheric CO2 concentrations ([CO2]) representing both modern (440 p.p.m. CO2) and ancient (1500 p.p.m. CO2) atmospheres.

Upon sporophyte maturation, stomata from each bryophyte species were imaged, measured and quantified. KEY RESULTS: Densities and dimensions were unaffected by changes in [CO2], other than a slight increase in stomatal density in Funaria and abnormalities in Polytrichum stomata under elevated [CO2].


The changes to stomata in Funaria and Polytrichum are attributed to differential growth of the sporophytes rather than stomata-specific responses. The absence of responses to changes in [CO2] in bryophytes is in line with findings previously reported in other early lineages of vascular plants.

These findings strengthen the hypothesis of an incremental acquisition of stomatal regulatory processes through land plant evolution and urge considerable caution in using stomatal densities as proxies for paleo-atmospheric CO2 concentrations.


Stomata of hornworts (Anthocerotophyta)

Photo credit: Google

Hornwort: Anthoceros punctatus

Stomatal differentiation and abnormal stomata in hornworts

by Pressel S., Goral T., Duckett J. G. (2014)

Silvia Pressel, Tomasz Goral

Jeffrey G. Duckett item21720

in Maney Online: Journal of Bryology, Volume 36, Issue 2 (June 2014), pp. 87-103

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.

See also: Maney Online