Stomata in mosses

XV. On the existence of stomata in mosses. In a Letter to Richard Horsman Solly, Esq., F.R.S. & L.S. 

Valentine W. (1839)

William Valentine,

Trans. Linn. Soc. 18: 239-245 –

Stomata of Bryophyta

Merced A. (xxxx) – Bryophyte Stomata –

Amelia Merced,

Stomata are present across all plants excluding liverworts and are very similar, consisting of a pore surrounded by two guard cells. In all extant plants, stomata are found in the sporophyte. The origin and evolution of stomata in land plants is controversial. Moss guard cells have similar wall architecture and are less variable than tracheophytes guard cells. In reduced sporophytes (such as Ephemerum and Physcomitrella) capsule anatomy is modified and some stomata features are also reduced (Merced & Renzaglia 2013). We described the developmental pattern and distribution of stomata in the moss Funaria (Merced & Renzaglia 2016) and changes in pectin composition during guard cell development. We found that guard cell walls are thinner and rich in pectins during the short period where stomata can open and close (Merced & Renzaglia 2014). We hypothesize that during development of the sporophyte, stomata serves as passages for gas exchange and bringing up water into the expanding capsule, later stomata helps to dry the capsule and influence the release of spores. The single origin of stomata is complicated by the absence of true stomata in early-divergent mosses, but Sphagnum has specialized epidermal cells, pseudostomata, that partially separate but do not form a pore. Pseudostomata may be related to stomata and share a common function to moss stomata (Merced 2015), with wall architecture and behavior specialized to facilitate capsule dehydration, shape change, and dehiscence. To have a better picture of stomata evolution we studied the ultrastructure, anatomy and composition of stomata of hornworts and proposed that they share a common architecture and fate to stomata of ancient plants (Renzaglia et al. 2017). It turns out that guard cell walls of hornworts lack some of the pectin components necessary for stomata movement that are present in angiosperms (Merced & Renzaglia 2019).
In a review article we summarize and synthesize the knowledge acquire in the last few years about bryophyte stomata and future directions of study (Merced & Renzaglia 2017).

Bryophyte Diversity And Ecology

Bryophytes are usually a neglected group of plants, being small they can be bypass without notice, but once you stop to look at them or better yet get ahold of a hand-lens or a microscope you will be able to see their beauty. Bryophytes is the collective name given to three groups of plants: mosses, liverworts and hornworts. I have been studying bryophytes since 2001.

I am working with the bryophytes of Puerto Rico, collecting and identifying bryophytes around the islands. In particular, focusing on the role of bryophytes in Puerto Rican forests and how they respond to anthropogenic and non-anthropogenic disturbances. I am also interested in urban and community forests that sustain bryophytes to understand how they are different to non-urban vegetations.

To better understand the distribution of bryophytes in a tropical forest, we are studying the presence and abundance of a common moss and liverwort in El Verde LTER. We are interested in learning what ecological factors influence the presence and size of these bryophytes, and how it compares to other plants.

Southern Illinois was a great area for bryophytes. In collaboration with S. Jesselson, a SIU Plant Biology undergraduate student in the Renzaglia lab, we collected, identify and image mosses of the area. Here is a link to the project of some common mosses of southern Illinois and the poster.

As a research assistant in Dr. Shaw’s Bryology Lab at Duke University I worked on the virtual flora of the mosses of North Carolina. The key to the mosses of NE United States, created by Lewis Anderson and others, is illustrated with pictures of some of the species and important characters for the identification. Here is the link to the Mosses of North Carolina.

From 2005 to 2008 I worked at the UPRRP Herbarium (San Juan PR), where I was in charge of the database activities of the herbarium and supervision of students. There I collected and identify specimens for the bryophyte collection. Here is the link to the UPRRP herbarium database.

During the time I was at UPR Río Piedras I worked with two undergraduate students doing research in bryology. With S. Galva we collected and identified bryophytes of the Carite Forest Reserve, PR and prepared a preliminary list of bryophyte species with new records for the area. Here is a poster with our findings (in Spanish). Working with orchid expert Dr. J. Ackerman I learn to see orchids everywhere. Did you know that small orchids, like Lepanthes, are often found between bryophyte matts? As an undergrad student J.G. García Cancel, advised by Dr. Melendez-Ackerman, looked at the relationship between orchid distribution and bryophyte cover, this study found that in thick bryophyte cover adult orchids are more frequent than younger plants but that interactions between bryophytes and this orchid are dynamic during different life stages. This research resulted in a publication in the Caribbean Naturalist.


Stomata of Funaria hygrometrica (Bryophyta)

The development and ultrastructure of the stomata of Funaria hygrometrica Hedw.

Sack F. D. (1982)

Fred David Sack,

Ph.D. thesis – Cornell University, Ithaca, New York –

Stomatal development in Funaria hygrometrica Hedw. was studied with light and electron microscopy. The stoma is one-celled at all stages. Nuclear division in the guard cell parent cell is followed by incomplete cytokinesis; the cell plate, and thus the newly formed septum, never reach the ends of the cell as they do in the stomata of most other plants. Preprophase bands of microtubules were absent in guard cell parent cells but present in some pre-division non-stomatal epidermal cells.

Throughout development, the guard cell wall is thinnest in areas of the outer and dorsal walls near the subsidiary cell and at the mid-depth of the ventral (pore) wall. The mature wall contains a mottled layer sandwiched between two more fibrillar layers. The internal wall layer has sublayers with fibrils in axial and radial orientations with respect to the pore. During substomatal cavity formation, the middle lamella is stretched into an electron dense network and into strands and sheets.

The rear and forechambers of the pore generally form before the central aperture and ledges do. Material present between the separating interfaces of the ventral wall is involved in cuticle assembly i.e. cuticle formation is simultaneous with the creation of the pore. This material includes rods, globules and a granular-fibrillar matrix. The contents of the globules form the bulk of the pore cuticle and the electron dense borders of the globules become the cuticular fibrils. Both the ventral and outer wall cuticles contain fibrils that sometimes reach the surface but the fibril arrangement is roughly perpendicular to the surface in the pore cuticle and reticulate in the outer wall cuticle. Fibrils are absent in the thinner, subsidiary cell cuticle.

Endoplasmic reticulum (ER) cisternae are initially rough and often arranged in parallel arrays. During pore formation, the cytoplasm becomes packed with tubular, smooth ER. Older but still functional stomata contain small amounts of primarily cisternal ER. Lipid bodies decrease in electron density when the tubular ER appears. Preliminary observations indicate that two large vacuoles occupy the polar regions of open but not closed stomata.

Reductive evolution of stomata

Phylogenomic evidence for reductive evolution of stomata

Leon J. (2020)

Jesus Leon,

(Curr. Biol.) – Plant Science Research Weekly  –

Colonization of the terrestrial environment by land plants (embryophytes), a monophyletic clade that evolved from freshwater streptophyte algae, forever changed Earth by transforming biogeochemical cycles. The evolution of stomata was a key adaptation that allowed the colonization of terra firma. Present in most land plants, stomata control the passage of carbon dioxide and water vapor, thus providing plants with a coping mechanism against arid and fluctuating conditions. The origin and ancestral function of stomata are obscure because of stomata-bearing and -lacking lineages and diversity in their morphology and function. By making use of new plant genomes and data from the 1KP project, Harris et al. sought to unravel the phylogeny of land plant and through it resolve the evolutionary history of stomatal development and function. By analyzing single-copy orthologs of 162 Viridiplantae genomes the authors determined that bryophytes were monophyletic and sister to tracheophytes, suggesting that the common ancestor of land plants already possessed stomata, with secondary loss of these in liverworts and some mosses. Subsequent presence/absence analyses of genes involved in stomata development and function confirmed this, as several genes essential for these processes were present in the common ancestor of embryophytes, whereas many of them were later lost in bryophytes. These results confirm bryophyte monophyly and shed light on stomata origin, suggesting that the ancestor of land plants possessed stomata, while bryophytes underwent reductive evolution probably due to loss of key developmental and functional regulators, with liverworts entirely losing stomata and later evolving liverwort-specific air pores. (Summary by Jesus Leon @jesussaur) Curr. Biol. 10.1016/j.cub.2020.03.048
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Significant correlations have been obtained between stoma number and seta length, and stoma size and epidermal cell size in mosses 

On the stomata of some tropical African mosses

Egunyomi A. (1982)

Lindbergia 8: 121–124 –


The occurrence of stomata in 29 tropical African moss species representing 12 families is reported. The stomata (22-51 μm × 22-29 μm) are mostly round-pored with two guard-cells each, ranging from 2 to more than 200 per capsule. In Wijkia trichocoleoides (C. Muell.) Crum, Trichosteleum microcalyx Ren. & Card., Stereophyllum radiculosum (Hook.) Mitt. and Stereophyllum virens Card. stomata are raised above the level of epidermis but are sunken in Brachymenium leptophyllum C. Muell.) Jaeg. and Bryum coronatum Schwaegr. Significant correlations have been obtained between stoma number and seta length, and stoma size and epidermal cell size. 

The morphological features of the stomata in ten moss species have not any taxonomical value

Observations on the stomatal complex in ten species of mosses (Pottiaceae, Bryopsida)

Abella L., Alcalde M., Estébanez B., Cortella A., Alfayate C., Ron E., (1999)

  • L. ABELLALaboratorio de Etnobotánica y Botánica Aplicada (L.E.B.A.), Facultad de Ciencias Naturales y Museo. Universidad Nacional de La Plata
  • M. ALCALDEDpto. Biología Vegetal I, Facultad de Biología, Universidad Complutense
  • B. ESTÉBANEZDpto. Biología Vegetal I, Facultad de Biología, Universidad Complutense
  • A. CORTELLALaboratorio de Etnobotánica y Botánica Aplicada (L.E.B.A.), Facultad de Ciencias Naturales y Museo. Universidad Nacional de La Plata
  • C. ALFAYATEDpto. Biología Vegetal I, Facultad de Biología, Universidad Complutense
  • E. RONDpto. Biología Vegetal I, Facultad de Biología, Universidad Complutense


J. Hattori Bot. Lab. 86: 179–185 – doi: 10.18968/jhbl.86.0_179


The stomatal complex in Bryophyta is morphologically more uniform than in vascular plants. Nevertheless there are many species without data about the morphology, physiology and ontogeny of their stomata. We present here a morphological and histochemical study of the stomatal complex in ten species of CrossidiumDidymodonPottia and Tortula (Pottiaceae).The number of stomata per capsule, their size, orientation, location, neighbouring cells, morphological type and the results of the histochemical tests for the guard and adjacent cells for starch, callose, cellulose and pectin are described. The results suggest that the morphological features of the stomata in these ten species have not any taxonomical value.

The anatomy of stomate and astomate taxa and the development of intercellular spaces, including substomatal cavities, across mosses

Figure 2. Stomata diversity in mosses. (A) Atrichum angustatum light micrograph of stomata free epidermis. (B) Funaria hygrometrica SEM of apophysis covered with ∼200 stomata. (C) Physcomitrium (Physcomitrella) patens 2 of 10 stomata in fluorescence. (D) Brachythecium rutabulum SEM of sparse scattered stomata. Image credit: Jeffrey J. Duckett. (E) Plagiomnium cuspidatum SEM showing numerous sunken stomata on the apophysis. 60 stomata estimated in the capsule. Image credit: Jeffrey J. Duckett. (F) Bartramia pomiforme group of stomata in fluorescence. 70 stomata estimated in the capsule. Bars: (A,C,F) = 20 μm, (B,D,E) = 50 μm.

With Over 60 Independent Losses, Stomata Are Expendable in Mosses

Renzaglia K. S., Browning W. B., Merced A. (2020)

Karen S. Renzaglia1*William B. Browning1 and Amelia Merced2

  • 1Plant Biology Department, Southern Illinois University, Carbondale, IL, United States
  • 2International Institute of Tropical Forestry, USDA Forest Service, San Juan, PR, United States


Front. Plant Sci., 28 May 2020 –

Figure 3. Capsule anatomy, pseudostomata and stomata in extant members of early divergent moss lineages, and sporangia and stomata of the first fossil land plants. (A) Takakia ceratophylla. Light micrograph (LM) longitudinal section of solid cylindrical capsule with spore mother cells (SM), columella (Co) and conducting strand (CS) in seta. (B) Andreaea rothii. LM longitudinal section of solid capsule with spores, columella (Co) and short seta (S) surrounded by gametophyte (G) tissue of the pseudopodium. (C) Sphagnum tenellum. LM longitudinal section of solid capsule, covered by calyptra (C), with pseudostomata (P) in the epidermis, massive columella (Co) covered by the spore sac, and highly reduced seta (S) embedded by foot (F) into gametophyte (G) pseudopodium. (D) Takakia ceratophylla capsule with single spiraled suture and spores. (E) Tortilicaulis transwalliensis capsule from the Silurian resembles Takakia in (D)(F) Sphagnum tenellum SEM showing scattered pseudostomata on dried capsule. (G) Early Devonian bivalved sporangium with scattered stomata (spots). (H) Early Devonian sporangium with band of stomata (spots) at base. (I) Oedipodium LM cross section of neck with guard cells with ledges over substomatal cavity. (J) Aglaophyton major from Rhynie Chert. Cross section of mature axis with stoma showing guard cells with ledges over substomatal cavity. Fossil images reproduced with permission from Journal of Experimental Botany (Edwards et al., 1998) and Paleontology (Edwards, 1979). Bars: (A,E,H) = 100 μm; (B,G,J) = 50 μm; (C,F) = 500 μm; (D)= 200 μm, (I) = 20 μm.

Because stomata in bryophytes are uniquely located on sporangia, the physiological and evolutionary constraints placed on bryophyte stomata are fundamentally different from those on leaves of tracheophytes. Although losses of stomata have been documented in mosses, the extent to which this evolutionary process occurred remains relatively unexplored. We initiated this study by plotting the known occurrences of stomata loss and numbers per capsule on the most recent moss phylogeny. From this, we identified 40 families and 74 genera that lack stomata, of which at least 63 are independent losses. No trends in stomata losses or numbers are evident in any direction across moss diversity. Extant taxa in early divergent moss lineages either lack stomata or produce pseudostomata that do not form pores. The earliest land plant macrofossils from 400 ma exhibit similar sporangial morphologies and stomatal distribution to extant mosses, suggesting that the earliest mosses may have possessed and lost stomata as is common in the group. To understand why stomata are expendable in mosses, we conducted comparative anatomical studies on a range of mosses with and without stomata. We compared the anatomy of stomate and astomate taxa and the development of intercellular spaces, including substomatal cavities, across mosses. Two types of intercellular spaces that develop differently are seen in peristomate mosses, those associated with stomata and those that surround the spore sac. Capsule architecture in astomate mosses ranges from solid in the taxa in early divergent lineages to containing an internal space that is directly connected to the conducing tissue and is involved in capsule expansion and the nourishment, hydration and development of spores. This anatomy reveals there are different architectural arrangements of tissues within moss capsules that are equally effective in accomplishing the essential processes of sporogenesis and spore dispersal. Stomata are not foundational to these processes.

Evidence for a less elaborated but effective mechanism for stomata spacing in plants

Mature capsules of Funaria. (A) Scanning electron micrograph of expanded capsule with stomata in irregular rows and files on apophysis (arrowheads). (B) Drawing of stomata distribution in the apophysis of mature capsule. (C, D) Scanning electron micrographs of spongy tissue inside the capsule. (E) Scanning electron micrograph of apophysis showing slightly raised stomata covered by smooth cuticle that is thickened around the pore (arrow). Scale bars: (A, C) = 500 µm; (B) = 35 µm; (D) = 100 µm; (E) = 10 µm.

Patterning of stomata in the moss Funaria: a simple way to space guard cells

Merced A., Renzaglia  K. S. (2016)

Amelia MercedKaren S. Renzaglia,

Ann. Bot. 117(6): 985–994 –

Early capsule expansion at the same stage as in Fig. 2D. (A) Predominantly longitudinal cell divisions of epidermal cells in the expanding apophysis as visualized by bright turquoise fluorescence of callose in new cell walls, detected by aniline blue. (B) Differential interference contrast image of capsule during expansion. Most stomata have differentiated and have ventral walls but no pore. Line drawing overlay of part of the capsule shows the arrangement of stomata. (C) Guard cells have abundant chloroplasts and are bigger than epidermal cells; stomata are arranged in files and rows. Distal round cells in the same files as stomata appear to be arrested stomata (arrowheads). (D) Aniline blue fluorescence of the same area as (C), identifying callose (bright turquoise) in newly formed walls of epidermal cells (arrows); divisions are mostly parallel to the sporophyte axis and are consistent with expansion in width of the capsule. Asterisks in C and D indicate the same stoma. (E) Light micrograph of capsule expansion with fully formed stomata with prominent peripheral chloroplasts. (F) Aniline blue fluorescence of the same area as in (E), showing callose (bright turquoise) in newly formed walls of epidermal cells in various planes around stomata (arrows). No callose is found in guard cell walls. Asterisks in (E) and (F) indicate the same stoma. Scale bars: (A) = 75 µm; (B, E, F) = 35µm; (C, D) = 10 µm.


Background and Aims Studies on stomatal development and the molecular mechanisms controlling patterning have provided new insights into cell signalling, cell fate determination and the evolution of these processes in plants. To fill a major gap in knowledge of stomatal patterning, this study describes the pattern of cell divisions that give rise to stomata and the underlying anatomical changes that occur during sporophyte development in the moss Funaria.

Methods Developing sporophytes at different stages were examined using light, fluorescence and electron microscopy; immunogold labelling was used to investigate the presence of pectin in the newly formed cavities.

Key Results Substomatal cavities are liquid-filled when formed and drying of spaces is synchronous with pore opening and capsule expansion. Stomata in mosses do not develop from a self-generating meristemoid as in Arabidopsis, but instead they originate from a protodermal cell that differentiates directly into a guard mother cell. Epidermal cells develop from protodermal or other epidermal cells, i.e. there are no stomatal lineage ground cells.

Conclusions Development of stomata in moss occurs by differentiation of guard mother cells arranged in files and spaced away from each other, and epidermal cells that continue to divide after stomata are formed. This research provides evidence for a less elaborated but effective mechanism for stomata spacing in plants, and we hypothesize that this operates by using some of the same core molecular signalling mechanism as angiosperms.

A genetic mechanism that is responsible for the development of stomata

This evolutionary innovation fundamentally changed the global cycles of carbon, water, and energy and thus was a prerequisite for all life forms on the mainland, including us humans.

Biologists discover origin of stomata

Chater C., Caine R. S., Tomek M., Wallace S., Kamisugi Y., Cuming A. C., Lang D., MacAlister C. A., Casson S., Bergmann D. C., Decker E. L., Frank W., Gray J. E., Fleming A., Reski R., Beerling D.J. (2016)

Caspar C. Chater, Robert S. Caine, Marta Tomek, Simon Wallace, Yasuko Kamisugi, Andrew C. Cuming, Daniel Lang, Cora A. MacAlister, Stuart Casson, Dominique C. Bergmann, Eva L. Decker, Wolfgang Frank, Julie E. Gray, Andrew Fleming, Ralf Reski, David J. Beerling,


Science Daily – Albert-Ludwigs-Universität Freiburg –


A similar genetic mechanism between flowering plants and mosses is a result of evolutionary conservation, reports an international team of researchers. They have discovered a genetic mechanism that is responsible for the development of stomata — microscopic valves on the surface of plants that facilitate the uptake of carbon dioxide and the release of oxygen and water vapor.


An international team has discovered a genetic mechanism that is responsible for the development of stomata — microscopic valves on the surface of plants that facilitate the uptake of carbon dioxide and the release of oxygen and water vapor. The researchers discovered this mechanism, which was previously known in flowering plants like Arabidopsis thaliana, in the moss Physcomitrella patens and found similarities between the two, implying that it already existed in the last common ancestor of mosses and flowering plants. The team was led by the biologists Professor Ralf Reski from the University of Freiburg/Germany and Professor David J. Beerling from the University of Sheffield/UK. The results were published in the journal Nature Plants.

Stomata came into being more than 400 million years ago when the first plants colonized the hitherto hostile land masses. Because stomata facilitate an efficient gas exchange with the atmosphere, they enabled the spread of plants and the subsequent evolution of our complex ecosystems. In contrast to more developed vascular plants with roots, stems, leaves, and vasculature, which are necessary for the transport of water and nutrients, it remained unclear in the case of mosses, which have no vasculature, which genes are responsible for the development of stomata.

The research team found that an interaction between the two proteins PpSMF1 and PpSCRM1 in Physcomitrella is the trigger responsible for the development of stomata in moss. When they deleted one of these genes, moss developed without stomata. The researchers found that this mechanism is similar to the interaction of the two proteins MUTE and FAMA, which triggers the development of stomata in Arabidopsis. The genes which encode these proteins therefore originate from the last common ancestor of mosses and flowering plants — the prehistoric plants which left the fresh water to dwell on rocks and thus laid the foundation for the development of all current ecosystems on the mainland.

“Our results show that the development of stomata originated over 400 million years ago and predated the development of roots, stems, and leaves,” explains Reski. “This evolutionary innovation fundamentally changed the global cycles of carbon, water, and energy and thus was a prerequisite for all life forms on the mainland, including us humans.”

Stomata of the Model Moss Physcomitrium patens

Figure 2. Stomatal development during sporophyte development in Physcomitrium patens(A–G) Overview of the developing P. patens sporophyte from fertilization to fully expanded brown sporophyte stage. (a–g) Close-ups of (A–G) illustrating early sporophyte development, and then once formed, stomata and their development in relation to overall sporophyte development. Representative stomata in panels (c–g) are marked with purple arrows. (A) Mature gametangia and nascent sporophyte (black arrow) surrounded by leafy gametophyte tissue. (a) Left image, a very young sporophyte (red arrow) resulting from the fertilization of the egg cell in the female archegonia. The gametophytic calyptra derived from the archegonia is visible and is being pushed up by the underlying nascent sporophyte. Right image, male antheridia (blue arrow) with a cloud of spermatozoids above. (B) Developing sporophyte (black arrow) being pushed up via a seta, with gametophyte calyptra still affixed (yellow arrow); the seta is subtending the calyptra. (b) A close-up of the calyptra sitting atop the gametophyte (yellow arrow). (C) Elongating sporophyte with a darkened central spore sac becoming visible. (c) Stomatal lineage cells protruding from the surface of epidermis (purple arrows). The calyptra is absent from this image, and also for subsequent images through to sporophyte maturity. Normally it remains present until the penultimate stages of sporophyte development when sporophytes remain undisturbed (Hiss et al., 2017). (D) As the sporophyte begins to expand outward the central spore sac becomes distinct from the surrounding tissue. (d) As expansion of the capsule occurs (D) stomatal pores can be seen in the central regions of recently formed guard cells (GCs; see centrally placed purple arrow). (E) As the central spore sac expands the overall shape of the sporophyte becomes more spherical. (e) The stomata on the expanding sporophyte begin a transition from being translucent to being filled with an orange to brown substance. (F) A fully expanded green sporophyte with maturing spores. (f) The GCs are now orange in color as the sporophyte is maturing. (G) The fully expanded sporophyte capsule is browned, indicating that the internal spores are mature. (g) Like the sporophyte capsule, the color of the stomata turns increasingly brown prior to and during senescence. (H) Scanning electron microscopy image of a mature Physcomitrium patens guard cell plugged with waxes. Scale bars are as follows: (A–G) = 100 μm; (a–g) = 50 μm; (H) = 25 μm.

Stomata and Sporophytes of the Model Moss Physcomitrium patens

Caine R., Chater C. C., Fleming A. J., Gray J. E. (2020)

Robert S. Caine1*†Caspar C. C. Chater1†Andrew J. Fleming2† and Julie E. Gray1†

  • 1Department of Molecular Biology and Biotechnology, University of Sheffield, Sheffield, United Kingdom
  • 2Department of Animal and Plant Sciences, University of Sheffield, Sheffield, United Kingdom


In Front. Plant Sci., 25 May 2020 –

Figure 3. Stomatal formation during capsule expansion and in fully expanded mature capsules. (A) A stacked and flattened image of an early expanding sporophyte capsule dissected and face-up. The capsules surveyed were equivalent in size to sporophytes including and between growth stages in Figures 2D,E(B) A stacked and flattened image of an equivalent fully expanded mature brown spore capsule equivalent to Figure 2G(C) Dot-plot of stomatal number on expanding and mature sporophyte capsules. Individual replicate values denoted by circles, means by black diamonds. A two-tailed t-test confirms significantly more stomata on mature brown capsules (∗∗P < 0.01). (D) A stomatal cluster consisting of two stomata (2-er) on a mature browned wild-type sporophyte capsule. (E) A stomatal cluster consisting of three stomata (3-er) on a mature wild-type capsule. (F) Dot-plot of instances of clustering on expanding and mature brown sporophyte capsules. Symbols are as in panel (C). Non-clustering stomata are counted as 1-ers. (G–N) All images taken from mature brown fixed capsules. (G) An epidermal area almost devoid of stomata. (H,I) Enlarged round, possibly aborted GMC cells (see black arrows). (J) An aborted GMC displaying the characteristic orange to brown hue akin to mature guard cells but without the central pore (see arrow). (K) Mature sporophyte capsule dissected and stained with diphenylboric acid 2-amino ethyl ester (DPBA). (L) Capsule exposed to UV light to assess flavonoid derivatives which are visible as an orange fluorescence in the guard cell. (M) Mock treated equivalent to panel (K)(N) No fluorescence is emitted from the guard cell under equivalent UV light treatment. Scales bars are as follows: (A,B) = 50 μm; (D,E) and (G–J) = 15 μm; (K–M) = 25 μm.


Mosses are an ancient land plant lineage and are therefore important in studying the evolution of plant developmental processes. Here, we describe stomatal development in the model moss species Physcomitrium patens (previously known as Physcomitrella patens) over the duration of sporophyte development. We dissect the molecular mechanisms guiding cell division and fate and highlight how stomatal function might vary under different environmental conditions. In contrast to the asymmetric entry divisions described in Arabidopsis thaliana, moss protodermal cells can enter the stomatal lineage directly by expanding into an oval shaped guard mother cell (GMC). We observed that when two early stage P. patens GMCs form adjacently, a spacing division can occur, leading to separation of the GMCs by an intervening epidermal spacer cell. We investigated whether orthologs of Arabidopsis stomatal development regulators are required for this spacing division. Our results indicated that bHLH transcription factors PpSMF1 and PpSCRM1 are required for GMC formation. Moreover, the ligand and receptor components PpEPF1 and PpTMM are also required for orientating cell divisions and preventing single or clustered early GMCs from developing adjacent to one another. The identification of GMC spacing divisions in P. patens raises the possibility that the ability to space stomatal lineage cells could have evolved before mosses diverged from the ancestral lineage. This would have enabled plants to integrate stomatal development with sporophyte growth and could underpin the adoption of multiple bHLH transcription factors and EPF ligands to more precisely control stomatal patterning in later diverging plant lineages. We also observed that when P. patens sporophyte capsules mature in wet conditions, stomata are typically plugged whereas under drier conditions this is not the case; instead, mucilage drying leads to hollow sub-stomatal cavities. This appears to aid capsule drying and provides further evidence for early land plant stomata contributing to capsule rupture and spore release.